Loss of WT1 expression in the endometrium of infertile PCOS patients: a hyperandrogenic effect?

CONTEXT: In fertile patients the endometrial Wilms tumor suppressor gene (WT1) is expressed during the window of implantation. Polycystic ovary syndrome (PCOS) patients suffer from hyperandrogenemia and infertility and have elevated endometrial androgen receptor (AR) expression. WT1 is known to be down-regulated by AR. Therefore, the expression of WT1 and its targets may be altered in PCOS endometrium. OBJECTIVE: The objective of the study was to assess the expression and regulation of WT1 and selected downstream targets in secretory endometrium from ovulatory PCOS (ovPCOS) and fertile women. DESIGN AND PATIENTS: Endometrial samples were obtained from 25 ovPCOS and 25 fertile patients. MAIN OUTCOME MEASURE: Endometrial expression of WT1 and selected downstream targets were assessed by immunohistochemistry and RT-PCR. The androgen effect on WT1 expression was determined in vitro by immunoblots and RT-PCR. The expression of WT1 and its targets was quantified in fertile and ovPCOS stromal cells in the presence of androgens by RT-PCR. Caspase-3/7 activity was measured to evaluate sensitivity to drug-induced apoptosis. RESULTS: WT1 expression was down-regulated in secretory-phase ovPCOS endometrium. Stromal expression of Bcl-2 and p27 was higher, and epidermal growth factor receptor was lower in ovPCOS than in fertile patients. Endometrial stromal expression of WT1, Bcl-2, Bcl-2-associated X protein, and ß-catenin was regulated by androgens. Apoptosis levels were reduced in ovPCOS samples and androgen-treated fertile samples. CONCLUSION: WT1 expression is down-regulated in ovPCOS endometrium during the window of implantation. Androgens regulate the expression of WT1 and its targets during endometrial decidualization. The altered balance between WT1 and AR in the endometrium of PCOS patients may jeopardize the success of decidualization and endometrial receptivity.

Cetrorelix in women with poor ovarian response in previous assisted reproduction cycles.

Our aim was to compare a gonadotrophin-releasing hormone (GnRH) antagonist protocol with an analogue protocol using high dose gonadotrophins (rFSH) in women with poor ovarian response in order to optimise the management while undergoing assisted reproduction treatment. We recruited 31 consecutive patients over 5 months. The eligibility criteria for the study were: one or more previous cancelled cycle due to or=4,500 IU of rFSH. For the antagonist cycle regimen, we used daily 300 IU of rFSH from day 2 on the menses, and then from day 5 daily 0.25 mg of Cetrorelix until the day of human chorionic gonadotrophin (hCG) administration. We demonstrated that the use of an antagonist cycle was associated with a reduction in cancellation rates from 48% (agonist) to 10% (antagonist) (p < 0.039) allowing women to undergoing oocyte retrieval and embryo transfer with a non-significant improvement in the pregnancy rates.

The effects of sex steroid hormones and interleukin-1-beta on MUC1 expression in endometrial epithelial cell lines.

Oestrogen, progesterone and paracrine signals from the embryo have been associated with the overall control of implantation. Changes in the expression of the heavily glycosylated transmembrane glycoprotein MUC1 mucin on the endometrial epithelium are also thought to be important for embryo attachment. Increased MUC1 expression has been correlated with elevated progesterone levels in the secretory phase of the menstrual cycle. Embryonic control of endometrial receptivity through changes in MUC1 expression could be achieved through the interleukin-1 system. Four endometrial epithelial cell lines (HEC1A, HEC1B, Ishikawa and RL592) were treated with oestrogen and progesterone (with or without interleukin-1-beta) and were subjected to immunocytochemistry and flow cytometric analysis to determine MUC1 production using MUC1 antibodies. HEC1A (oestrogen receptor (ER) and progesterone receptor (PR) positive) and HEC1B (ER positive and PR negative) were transfected with the MUC1 promoter, underwent similar treatment regimes and the activity of the MUC1 promoter relative to their untreated controls was determined using a chloramphenicol acetyltransferase (CAT) enzyme-linked immunoassay. Using the cell lines, we determined that endometrial MUC1 expression is up-regulated by progesterone, consistent with the in vivo increases in MUC1 related to high progesterone levels. We also revealed that neither oestrogen, nor interleukin-1-beta, appear to modulate MUC1. Progesterone-dependent regulation of MUC1 is likely to be an important factor in determining endometrial receptivity.

The expression pattern of MUC1 glycoforms and other biomarkers of endometrial receptivity in fertile and infertile women.

Changes in the surface epithelium of the endometrium, characterized in part by alterations in cell-surface molecules, sex steroid receptors and the appearance of pinopodes, coincide with the window of endometrial receptivity in the menstrual cycle. This study was performed to evaluate the usefulness of hematoxylin and eosin staining, scanning and transmission microscopy, and MUC1 glycoform, sex steroid receptor, and interleukin receptor (type 1) expression as biomarkers of endometrial receptivity using carefully characterized clinical fertile and infertile groups of women. Using a combination of immunohistochemistry and scanning electron microscopy (SEM) called scanning immunoelectron microscopy (SIM), we confirmed that MUC1 mucin was not associated with the endometrial pinopodes, which have been linked with embryo adhesion. We also showed that failure of embryo implantation was associated with an abnormal endometrial expression of MUC1 mucin, and retention of nuclear progesterone receptor (PR) particularly in epithelial cells. Hematoxylin and eosin staining, transmission electron microscopy (TEM), SEM in isolation and immunohistochemistry for interleukin receptor were not shown to be useful markers. Progesterone-dependent regulation of MUC1 appears to be an important factor in determining endometrial receptivity.

Day 14 maternal serum progesterone levels predict pregnancy outcome in IVF/ICSI treatment cycles: a prospective study.

BACKGROUND: Serum progesterone has been advocated as a tool in the diagnosis of early pregnancy failure. We conducted this prospective study in order to investigate the potential value of early (14 days after oocyte recovery) serum progesterone measurement, in women undergoing IVF/ICSI and receiving rectal progesterone supplements, in relation to pregnancy outcome. METHODS: 442 women consecutively treated by IVF or ICSI had serum progesterone and bhCG levels prospectively measured 14 days after oocyte retrieval (day 0). All women received natural progesterone 400 mg rectally until the pregnancy test on day 14. Pregnant women were followed up by serial transvaginal ultrasound scans to 8 weeks gestation. RESULTS: 115 women (26%) had a viable intra-uterine pregnancy at 8 weeks gestation, 80 (18.1%) had an abnormal pregnancy (biochemical, ectopic, miscarriage) and 247 (55.9%) failed to conceive. Women with on-going pregnancies had significantly higher serum progesterone levels (median: 430, 95%CI: 390-500 nmol/l) compared to those who had either an abnormal pregnancy (72, 48-96 nmol/l; P < 0.001) or failed to conceive (33, 28-37 nmol/l; P < 0.001). Receiver-operator curve analysis demonstrated that a single serum progesterone on day 14 post-oocyte retrieval, could highly differentiate between normal and abnormal pregnancies (area under the curve = 0.927, 95%CI = 0.89-0.96; P < 0.0001). CONCLUSIONS: In spite of exogenous progesterone supplementation, serum progesterone levels, from as early as 4 weeks gestation (day 14 post-oocyte retrieval) were significantly elevated and predicted women destined to have viable intra-uterine pregnancies. These high levels are suggestive that endogenous progesterone is already sufficient in viable pregnancies and that exogenous progesterone administration will not rescue a pregnancy destined to result in a miscarriage. Single serum progesterone measurement could be a useful indicator of pregnancy outcome in women undergoing IVF or ICSI treatment.

Impaired insulin-dependent glucose metabolism in granulosa-lutein cells from anovulatory women with polycystic ovaries.

BACKGROUND: Insulin resistance and hyperinsulinaemia are well-recognized characteristics of anovulatory women with polycystic ovary syndrome (PCOS) but, paradoxically, steroidogenesis by PCOS granulosa cells remains responsive to insulin. The hypothesis to be tested in this study is that insulin resistance in the ovary is confined to the metabolic effects of insulin (i.e. glucose uptake and metabolism), whereas the steroidogenic action of insulin remains intact. METHODS: Granulosa-lutein cells were obtained during IVF cycles from seven women with normal ovaries, six ovulatory women with PCO (ovPCO) and seven anovulatory women with PCO (anovPCO). Mean body mass index was in the normal range in all three groups. Granulosa-lutein cells were cultured with insulin (1, 10, 100 and 1000 ng/ml) and LH (1, 2.5 and 5 ng/ml). Media were sampled at 24 and 48 h and analysed for glucose uptake, lactate production and (48 h only) progesterone production. RESULTS: Insulin-stimulated glucose uptake by cells from anovPCO was attenuated at higher doses of insulin (100 and 1000 ng/ml) compared with that by cells from either ovPCO (P=0.02) or controls (P=0.02). Insulin and LH stimulated lactate production in a dose-dependent manner, but insulin-dependent lactate production was markedly impaired in granulosa-lutein cells from anovPCO compared with either normal (P=0.002) or ovPCO (P<0.0001). By contrast, there was no difference in insulin-stimulated progesterone production between granulosa-lutein cells from the three ovarian types. CONCLUSIONS: Granulosa-lutein cells from women with anovPCOS are relatively resistant to the effects of insulin-stimulated glucose uptake and utilization compared with those from normal and ovPCO, whilst maintaining normal steroidogenic output in response to physiological doses of insulin. These studies support the probability of a post-receptor, signalling pathway-specific impairment of insulin action in PCOS.

Formation and early development of follicles in the polycystic ovary.

BACKGROUND: Polycystic ovary syndrome is the most common cause of anovulatory infertility. It has long-term health implications and is an important risk factor for type 2 diabetes. However, little is known about the cause of polycystic ovaries. We have used detailed morphological analysis to assess the hypothesis that there is an intrinsic ovarian abnormality that affects the earliest stages of follicular development. METHODS: We took small cortical biopsies during routine laparoscopy from 24 women with normal ovaries and regular cycles and from 32 women with polycystic ovaries, 16 of whom had regular, ovulatory cycles and 16 of whom had oligomenorrhoea. We used computerised image analysis to assess the density and developmental stage of small preantral follicles in serial sections of fixed tissue. FINDINGS: Median density of small preantral follicles, including those at primordial and primary stages, was six-fold greater in biopsies from polycystic ovaries in anovulatory women than in normal ovaries (p=0.009). In both ovulatory and anovulatory women with polycystic ovaries, we noted a significant increase in the percentage of early growing (primary) follicles and a reciprocal decrease in the proportion of primordial follicles compared with normal ovaries. INTERPRETATION: Our findings indicate that there are fundamental differences between polycystic and normal ovaries in early follicular development, suggesting an intrinsic ovarian abnormality. The increased density of small preantral follicles in polycystic ovaries could result from increased population of the fetal ovary by germ cells, or from decreased rate of loss of oocytes during late gestation, childhood, and puberty.

Is there a link between an extremely poor response to ovarian hyperstimulation and early ovarian failure?

BACKGROUND: It has been previously reported that a group of 12 infertile women, who had a normal baseline hormonal profile and did not respond to repeated ovarian stimulation with gonadotrophins, developed ovarian failure within a few months. Based on this observation, we carried out a controlled retrospective cohort study to examine whether non-response to ovarian stimulation is linked to early ovarian failure. METHODS: All patients aged 35-40 years who had cancelled IVF cycles for non-response between 1991 and 1993 in our centre were asked to report on the subsequent development of menopausal symptoms, menopause or commencement of hormone replacement therapy. A control group consisted of patients with the same age and similar medical history, who had IVF the same year and responded well. RESULTS: Eleven out of the 12 patients of the non-response group developed menopausal symptoms within 7 years, compared with only four out of 24 in the control group. Similarly, eight out of 12 non-responders either went into menopause or started using hormone replacement therapy compared with one out of 24 in the control group. Using Fisher's exact test, the differences were highly significant (P < 0.0001). The median age at development of menopausal symptoms in the study group was 40 years (range 38-45). The median time between non-response and development of menopausal symptoms was 4 years (range 1-7). CONCLUSION: We carried out a controlled retrospective cohort study that showed a strong association between an extremely poor response to ovarian hyperstimulation and early ovarian failure.

Expression of Wilms' tumor suppressor gene (WT1) in human endometrium: regulation through decidual differentiation.

The Wilms' tumor suppressor gene (WT1) encodes a zinc-finger containing transcription factor that is selectively expressed in the developing urogenital tract and functions as a tissue-specific developmental regulator. In addition to its gene-regulatory function through DNA binding properties, WT-1 also regulates transcription by formation of protein-protein complexes. These properties place WT-1 as a major regulator of cell growth and differentiation. In view of these observations, we studied WT1 mRNA and protein in human endometrial extracts and in endometrial stromal cells (ESCs) differentiating into decidual cells in vitro, by RT-PCR and Western blotting, respectively. WT1 protein expression was also studied in situ in the proliferative and the secretory phase of the menstrual cycle in the early pregnant state. Analysis by PCR of total RNA prepared from human ESCs demonstrated the presence of WT1 mRNA and four WT1 mRNA splice variants. Western blot analysis of nuclear protein extracts from ESCs yielded one immunoreactive protein of the expected size (approximately 52-54 kDa) recognized by the WT1 antibody. Immunohistochemical staining showed that WT1 protein is localized only to nuclei of human endometrial stromal cells. It remains constant in the proliferative and the secretory phase of the menstrual cycle and is increased remarkably during decidualization in early pregnancy. ESCs decidualized in vitro were investigated for WT-1 expression, which confirmed that decidualizing stimuli (E2, medroxy-progesterone-acetate, and relaxin for 12 d or cAMP and progesterone for 1-4 d) induced WT-1 mRNA (P < 0.05) and increased protein levels (P < 0.05). These data indicate that in humans the WT1 gene is expressed in ESCs and its mRNA and protein levels remain constant in the proliferative and the secretory phase of the menstrual cycle and that WT1 mRNA and protein expression increases significantly in ESCs when these cells differentiate into decidual cells.

MUC 1: a genetic susceptibility to infertility?

In man and some animals regulation of embryo implantation by endometrial expression of the highly polymorphic MUC 1 mucin has been suggested. We assessed the polymorphism of MUC 1 in women known to be fertile and those with infertility due to suspected failure of embryo implantation. The median of the lower allele size in the infertile group was only 2.5 kb compared with 3.4 kb in the fertile group (p=0.0029, difference 0.9, [95% CI 0.1-1.3]). Women with unexplained infertility might have a genetic susceptibility to failure of embryo implantation due to small MUC 1 allele size.

Clinical experience with recombinant follicle-stimulating hormone (FSH) and urinary FSH: a retrospective case- controlled analysis.

OBJECTIVE: To compare the efficacy and efficiency of recombinant FSH (rFSH) and urinary FSH (uFSH). DESIGN: Retrospective case controlled analysis. SETTING: An assisted reproduction unit at a university center. PATIENT(S): 1388 patients undergoing long protocol in vitro fertilization/embryo transfer (IVF-ET) using buserelin acetate from day 2 of the cycle and either rFSH (follitropin beta) (n = 694) or uFSH (n = 694) with equal number of ampules started (rFSH: 50 IU, uFSH: 75 IU). INTERVENTION(S): Patients were included in the two groups of treatment after matching for similarity in age and type of treatment (IVF or intracytoplasmic sperm injection). MAIN OUTCOME MEASURE(S): Total dose of FSH, ovarian response, and IVF outcome. RESULT(S): Patients who received uFSH experienced a shorter period of stimulation, and a higher number of oocytes were collected. The total FSH used was lower in the rFSH group, and they required a lower FSH dose per oocyte retrieved. The implantation and pregnancy rates were similar between the uFSH and rFSH groups. In both groups implantation and pregnancy rates were higher when intracytoplasmic sperm injection was performed as compared with IVF. CONCLUSION(S): The implantation and pregnancy rates are similar when either rFSH or uFSH is used (when compared on an ampule-to-ampule basis, rFSH: 50 IU, and uFSH: 75 IU). However, a significantly lower total FSH dose was used in the rFSH group with a lower FSH dose per oocyte collected.

Dynamic assays of inhibin B and oestradiol following buserelin acetate administration as predictors of ovarian response in IVF.

The study was designed to examine whether dynamic measurements of inhibin B and oestradiol following single administration of buserelin acetate were correlated with the ovarian response to stimulation in IVF. A total of 37 patients undergoing IVF treatment was studied when the long protocol was started in the early follicular phase. Blood samples were taken twice: on day 2 of the menstrual cycle, before the first s.c. administration of buserelin acetate 0.5 mg and on day 3, 24 h later. Inhibin B and oestradiol concentrations were compared with the ovarian response to stimulation. The ovarian response was defined in two ways: 'number of oocytes/total recombinant (r) follicle stimulating hormone (FSH) dose'; and 'square-root (number of follicles/total rFSH dose)'. The following measurements were highly correlated with the ovarian response to stimulation: increase in oestradiol (day 3-day 2 oestradiol concentration) [correlation coefficient (r) = 0.68, P: < 0.0001] and sum of inhibin B (day 2 + day 3 inhibin B concentrations) (r = 0.6, P: < 0.0001). Age and basal concentrations of FSH and inhibin B were inferior to the above measurements in terms of correlation with the ovarian response. In conclusion, dynamic measurements of inhibin B and oestradiol following single administration of buserelin acetate were highly correlated with the ovarian response to stimulation for IVF treatment.

Pregnancy outcome is not affected by antiphospholipid antibody status in women referred for in vitro fertilization.

OBJECTIVE: To determine the prevalence of antiphospholipid (aPL) and anti-beta 2 glycoprotein I (anti-beta2-GPI) antibodies in women referred for IVF and to prospectively evaluate the effect of these antibodies on IVF outcome. DESIGN: Prospective observational study. SETTING: A university hospital and IVF unit. PATIENT(S): Three hundred eighty consecutive women referred for IVF. INTERVENTION(S): Blood samples taken before commencement of IVF cycles were tested for the presence of aPL (lupus anticoagulant [LA], anticardiolipin [aCL], and antiphosphatidyl serine antibodies [aPS]) and anti-beta2-GPI antibodies. MAIN OUTCOME MEASURE(S): Antibody prevalence, pregnancy rates, and live birth rates. RESULT(S): Of the total 380 women, 89 tested persistently positive for aPL (23.4%). None of 176 women tested for IgG aPS antibodies had a positive titer. Only 3.3% (11 of 329) tested positive for anti-beta2-GPI antibodies. Pregnancy rate, live birth rate, gestational age at delivery, and birth weight were not affected by aPL status. CONCLUSION(S): Although women referred for IVF have a high prevalence of aPL, these antibodies do not affect the outcome of treatment. Screening women undergoing IVF for aPL is not justified.

The significance of delayed suppression using buserelin acetate and recombinant follicle-stimulating hormone in a long protocol in vitro fertilization program.

OBJECTIVE: To determine whether the time taken to achieve ovarian suppression has an impact on ovarian responsiveness and the outcome of IVF-ET. DESIGN: Retrospective analysis. SETTING: An assisted reproduction unit at a university center. PATIENT(S): Patients undergoing a long protocol of IVF-ET that included buserelin acetate therapy initiated on day 2 of the cycle and recombinant FSH. INTERVENTION(S): Patients were divided into two groups according to the duration of buserelin acetate therapy required to achieve pituitary and ovarian suppression (group 1 = 2 weeks, n = 172; group 2 = > or =3 weeks, n = 337). MAIN OUTCOME MEASURE(S): Number of recombinant FSH ampules administered, duration of ovarian stimulation (days), ovarian response, and IVF outcome. RESULT(S): The patients in group 2 had lower mean E2 levels after 5 days and 9 days of stimulation than the patients in group 1. The number of recombinant FSH ampules administered and the number of days of stimulation required were higher in group 2 than in group 1. These differences were prominent in the subgroups of older patients (> or =36 years) and patients who had no evidence of polycystic ovaries on ultrasound examination. The number of oocytes retrieved and fertilized, the cancelation rate, and the pregnancy rate were similar in the two groups. CONCLUSION(S): Prolonged administration of a GnRH agonist to achieve suppression leads to a reduced ovarian response, particularly in women > or =36 years of age, but does not affect the success rate of IVF-ET.

Effects of follicle-stimulating hormone and serum substitution on the in-vitro growth of human ovarian follicles.

In-vitro maturation (IVM) of human ovarian follicles and oocytes could benefit infertile women, and allow the development of in-vitro systems for the study of human follicular development. Little is known about the initiation of growth of primordial follicles and the regulation of early folliculogenesis. An ovarian tissue-slice culture system was used to examine the effects of media composition, follicle stimulating hormone (FSH) and serum substitution on the development of small human follicles in vitro. Human ovarian cortex biopsies were cut into small pieces and cultured for 5, 10 or 15 days. Control (non-cultured) and cultured tissue was fixed, serially sectioned, and stained. The follicles contained within the tissue pieces were counted, measured, and assessed for stage of development and viability. Comparison of the ability of alpha-minimum essential medium (alpha-MEM), Waymouth's, or Earle's balanced salt solution (EBSS) culture media (all with 10% human serum) to support follicle growth demonstrated significantly increased initiation and growth of follicles in alpha-MEM during the first 10 days of culture. The supplementation of alpha-MEM with 300 mIU/ml FSH significantly reduced levels of atresia and increased the mean diameter of healthy follicles. Follicles in tissue cultured for 10 days with human serum albumin and ITS (insulin/transferrin/selenium mix) were significantly larger, more developed and showed significantly less atresia than those cultured with serum alone. Primordial to small preantral follicles can be grown under serum-substituted conditions in tissue-slice culture, and are responsive to FSH, which is thought to be acting mainly as a survival factor at these early stages.

One thousand initiated cycles of in vitro fertilization in women > or = 40 years of age.

OBJECTIVE: To evaluate the results of IVF in women > or = 40 years of age using their own oocytes. DESIGN: Retrospective study. SETTING: Wolfson and Royal Masonic in vitro fertilization units, London, United Kingdom. PATIENT(S): A total of 1,087 IVF cycles were started in women > or = 40 years of age. INTERVENTION(S): Medical records of patient outcomes were reviewed. MAIN OUTCOME MEASURE(S): Clinical pregnancy, miscarriage, and delivery rates. RESULT(S): Of the 1,087 cycles started in 471 women > or = 40 years of age, 842 reached oocyte retrieval (77.5%) and 702 had embryos transferred (64.6%). The pregnancy rate (PR) was significantly lower in women > or = 40 years of age than in a control group of women <40 years of age (11.3% versus 28.2%). It decreased sharply in women >42 years of age, and no women >45 years of age had a child. Women > or = 40 years of age were more likely to miscarry (27% versus 12.7%). When only one embryo was available for transfer, the PR was 3.3%. When >2 embryos were available for transfer, the PR was similar whether 2 or 3 embryos were replaced. No triplet pregnancy occurred. Women > or = 40 years of age achieved a cumulative PR of 30% after three cycles with a cumulative "take home baby" rate of 21%. CONCLUSION(S): In vitro fertilization is a reasonable treatment for women <45 years of age using their own gametes. Those with a "good response" in their first attempt may be encouraged to complete three cycles with an acceptable chance of conception.

Effect of salpingectomy on ovarian response to superovulation in an in vitro fertilization-embryo transfer program.

OBJECTIVE: To investigate the effect of salpingectomy on the response of each ovary in patients undergoing an IVF-ET treatment cycle and to compare the results with those of patients who had not had surgery and were undergoing IVF-ET during the same period. DESIGN: A prospective study. SETTING: Tertiary referral academic IVF unit. PATIENT(S): Twenty-nine ET cycles were evaluated in 29 patients who previously had undergone unilateral salpingectomy because of ectopic pregnancy (study group). Seventy-three patients with unexplained or male factor infertility served as controls. INTERVENTION(S): Ovulation induction and IVF-ET. MAIN OUTCOME MEASURE(S): In the study group, mean ovarian volume, number of follicles, and number of oocytes recovered from each ovary were assessed and compared. The overall results, cycle characteristics, and pregnancy rates of the two groups were compared. RESULT(S): Among the patients who had undergone salpingectomy, significantly fewer follicles developed and consequently fewer oocytes were retrieved from the ovary on the operated side (4.4 versus 8.2 follicles and 3.8 versus 6.0 oocytes). There were no differences in the total numbers of follicles and oocytes recovered from both ovaries, the cycle characteristics, or the pregnancy rates between study and control groups. CONCLUSION(S): Salpingectomy has no detrimental effect on the total ovarian performance during IVF-ET treatment or on the outcome of IVF-ET. However, the ipsilateral ovary could be adversely affected. This could be detrimental in selected patients undergoing IVF-ET, in whom the second ovary already is compromised or missing.

Insulin sensitivity in women at risk of coronary heart disease and the effect of a low glycemic diet.

The risk of coronary heart disease (CHD) is influenced by family history, insulin sensitivity (IS), and diet. Adiposity affects CHD and IS. The cellular mechanism of IS is thought to involve the adipocyte cytokine tumor necrosis factor-alpha (TNF-alpha). Insulin-stimulated glucose uptake in isolated subcutaneous and omental adipocytes obtained during elective surgery was measured in 61 premenopausal women, 24 with a parental history (PH) of CHD. In vivo IS was measured using the short insulin tolerance test (SITT) in 28 women, 16 with PH-CHD, before and 3 weeks after randomization to a low glycemic index (LGI) or high glycemic index (HGI) diet. In vitro adipocyte IS and TNF-alpha production was measured following dietary modification. On the habitual diet, in vitro insulin-stimulated glucose uptake in adipocytes as a percentage increase over basal was less in women with PH-CHD than in those without it (presented as the median with 95% confidence limits: subcutaneous, 28% (17% to 39%) v 96% (70% to 120%), P < .01); omental, 40% (28% to 52%) v 113% (83% to 143%), P < .01). In vivo IS in 16 PH-CHD subjects and 12 controls before dietary randomization was similar, and increased in both groups consuming a LGI versus HGI diet (PH-CHD, 0.31 (0.26 to 0.37) v 0.14 (0.10 to 0.24) mmol/L/min, P < .01; controls, 0.31 (0.1 to 0.53) v 0.15 (0.06 to 0.23) mmol/L/min, P < .05). Adipocyte IS was greater in PH-CHD women on a LGI versus HGI diet (subcutaneous, 50% (20% to 98%) v 13% (1% to 29%); omental, 97% (47% to 184%) v 29% (4% to 84%), P < .05). Adipocyte TNF-alpha production was higher in women with versus without PH-CHD (subcutaneous, 0.3 (0.18 to 0.42) v 0.93 (0.39 to 1.30) ng/mL/min; visceral, 0.22 (0.15 to 1.30) v 0.64 (0.24 to 1.1) ng/mL/min, P < .04, respectively), but was uninfluenced by the dietary glycemic index. We conclude that in vitro adipocyte IS is reduced and adipocyte TNF-alpha production is increased in premenopausal women with PH-CHD. A LGI diet improves both adipocyte IS in women with PH-CHD and in vivo IS in women with and without PH-CHD.