Douglas R. Danforth

Women with declining ovarian reserve may demonstrate a decrease in day 3 serum inhibin B before a rise in day 3 follicle-stimulating hormone☆

OBJECTIVE To test the hypothesis that women with declining ovarian reserve may demonstrate a decrease in day 3 serum inhibin B levels before a rise in day 3 serum FSH levels. DESIGN Case-control study. SETTING Tertiary care fertility center. PATIENT(S) One hundred nine women with nonovarian infertility (tubal factor or male factor) and 47 women with declining ovarian reserve who underwent assisted reproductive techniques. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Serum inhibin B and FSH levels, number of ampules of gonadotropins administered, E2 levels on the day of hCG administration, number of oocytes retrieved, clinical pregnancy rate, and cycle cancellation rate. RESULT(S) Women who had declining ovarian reserve as demonstrated by an increased gonadotropin requirement, a decreased E2 response, fewer retrieved oocytes, a lower clinical pregnancy rate, and a higher cycle cancellation rate had lower day 3 serum inhibin B levels despite having nonelevated day 3 FSH levels similar to those of women with nonovarian infertility. CONCLUSION(S) Women with declining ovarian responsiveness and clinical outcomes consistent with declining ovarian reserve had decreased day 3 serum inhibin B levels despite having nonelevated day 3 serum FSH concentrations. Declining ovarian reserve may be demonstrated by a decrease in day 3 inhibin B levels before a rise in day 3 FSH levels.

Vascular endothelial growth factor, nitric oxide, and leptin follicular fluid levels correlate negatively with embryo quality in IVF patients

OBJECTIVE(S) To measure vascular endothelial growth factor (VEGF), nitric oxide (NO) and leptin levels in individual ovarian follicles and to examine their relationships with perifollicular blood flow, follicular metabolic indices, and the developmental potential of the corresponding oocyte and embryo. DESIGN Prospective study. SETTING Academic, tertiary care institution. PATIENT(S) Unselected IVF patients. INTERVENTION(S) Color-pulsed Doppler analysis of perifollicular blood flow; determination of partial pressure of oxygen (pO2), partial pressure of carbon dioxide (pCO2), and pH and VEGF, leptin and NO levels in follicular fluid. MAIN OUTCOME MEASURE(S) Fertilization and day 3 embryo morphology and cleavage. RESULT(S) Fifty-five follicular fluid samples from 16 patients were studied. Mean follicular fluid levels were as follows: VEGF, 1,046+/-863.7 pg/mL (range, <63-3,332.7 pg/mL); NO3/NO2, 34.2+/-12 microM (range, 16.4-76.1 microM); and leptin, 20.1+/-12.1 ng/mL (range, 3.3-52.2 ng/mL). Vascular endothelial growth factor had a negative correlation with embryo morphology (r = -0.28, P = .01). Leptin demonstrated a negative correlation with follicular pO2 (r = -0.42, P = .005) and a positive correlation with follicular pCO2 (r = 0.36, P = .02). Follicular leptin levels correlated positively with VEGF levels (r = 0.46, P = .008) and with NO3/NO2 levels (r = 0.39, P =.006). CONCLUSION(S) Vascular endothelial growth factor, NO and leptin appear to be markers of follicular hypoxia and suboptimal embryo development. Whether fluctuations of these regulatory factors determine or reflect changes in the follicular microenvironment affecting oocyte developmental potential remains to be elucidated.

Follicular fluid vascular endothelial growth factor concentrations are elevated in women of advanced reproductive age undergoing ovulation induction

OBJECTIVE(S) To determine whether follicular fluid (FF) from women of advanced reproductive age had a relative deficiency of the angiogenic cytokine vascular endothelial growth factor/vascular permeability factor. Furthermore, we sought to determine whether luteinized granulosa cells secrete vascular endothelial growth factor/vascular permeability factor in response to hypoxia. DESIGN Retrospective cohort study. SETTING University teaching hospital. PATIENTS Women undergoing follicular aspiration after superovulation in preparation for IVF-ET. Women of advanced reproductive age consisted of 21 women > or = 38 years old (range, 38 to 46 years); 15 subjects < or = 30 years served as the control population. INTERVENTION(S) Granulosa cells and FF were collected by transvaginal aspiration 35 hours after hCG. Granulosa cells from two women were cultured for 24 and 48 hours in M199 + 10% fetal bovine serum in 1% O2-5% CO2-94% N2 (hypoxic) or 95% air-5% CO2 (normoxic) without or with 0.1 mol/L cobalt chloride. MAIN OUTCOME MEASURE(S) Pooled FF vascular endothelial growth factor/vascular permeability factor concentrations and media vascular endothelial growth factor/vascular permeability factor accumulation at 24 and 48 hours were determined. RESULT(S) Follicular fluid vascular endothelial growth factor/vascular permeability factor concentrations were higher in advanced reproductive age women compared with younger women (3,735 +/- 2,155 versus 2,205 +/- 952 pg/mL, mean +/- SD). Accumulation of vascular endothelial growth factor/vascular permeability factor at 24 and 48 hours was 391 +/- 54 and 744 +/- 2 pg/mL in media maintained in 5% CO2 and air. Cobalt chloride induced a marked increase in vascular endothelial growth factor/vascular permeability factor (2,008 +/- 52 pg/mL at 24 hours and 3,630 +/- 519 pg/mL at 48 hours). An intermediate but significant increase in vascular endothelial growth factor/vascular permeability factor (733 +/- 35 pg/mL at 24 hours and 2,675 +/- 864 pg/mL at 48 hours) was observed with 1% O2 compared with normoxic controls. CONCLUSION(S) After hMG and hCG administration the FF from women of advanced reproductive age showed increased vascular endothelial growth factor/vascular permeability factor concentrations compared with younger women. Increased vascular endothelial growth factor/vascular permeability factor concentrations could be consistent with a hypoxic environment within follicles of older women.

Vascular Endothelial Growth Factor Stimulates Preantral Follicle Growth in the Rat Ovary

Abstract The regulation of preantral follicle growth in mammals is poorly understood. The availability of an adequate vascular supply to provide endocrine and paracrine signals may be important during the early states of follicle growth as well as the later states of follicle selection and dominance. The objective of the present study was to investigate whether vascular endothelial growth factor (VEGF) plays a role in preantral follicular development in the rat ovary. Immature (age, 21 days) Sprague-Dawley rats were injected with 500 ng of VEGF in saline or 50 μg of diethylstilbestrol (DES) in oil under the bursa of one ovary. The contralateral ovary was injected with a corresponding volume of vehicle. Rats were killed 48 h later, and the ovaries were removed and analyzed histologically. Intrabursal administration of VEGF significantly increased the number of primary and small secondary, but not of large secondary, preantral follicles in the ovary, similar to the effect of DES (P < 0.05). The VEGF stimulated preantral follicle growth in a time- and dose-dependent manner. Subcutaneous DES administration increased the number of primary and secondary follicles, and both s.c. and intrabursal estrogen administration stimulated VEGF protein expression in the rat ovary. These data indicate that VEGF stimulates preantral follicular development in the rat ovary, is regulated by estrogen, and may be one of the factors that participate in the regulation of early follicle growth in the rat.

Ovarian control of follicle development.

A variety of ovarian autocrine and paracrine factors may modulate folliculogenesis and steroid production. The developmental program that leads to the production of a dominant follicle involves a precise quantitative and temporal pattern of expression of a large number of genes. Follicle-stimulating hormone plays an essential role in this process, and no other ligand by itself can serve in this regulatory capacity. It is clear that a variety of growth factors can modulate follicle-stimulating hormone action by autocrine and paracrine mechanisms. Advances in the understanding of the role of growth factors, particularly the family of insulin-like growth factor-related proteins, in regulating follicle-stimulating hormone action are discussed. It is likely that complex interactions exist between follicle-stimulating hormone and the growth factors. Significantly, growth factor regulation by pituitary gonadotropins is probably a central feature of their expression. With increased understanding of the ovarian control of follicle development, it is hoped that newer and more effective regimens for synchronous follicular and oocyte maturation can be realized.

Inhibin-B : the physiologic basis of the clomiphene citrate challenge test for ovarian reserve screening

OBJECTIVE To determine inhibin-B concentrations during ovarian reserve screening in women with normal and diminished ovarian reserve as determined by the clomiphene citrate challenge test. DESIGN Retrospective. SETTING Tertiary fertility center. PATIENT(S) Women undergoing ovarian reserve screening for a routine fertility evaluation. INTERVENTION(S) Clomiphene citrate challenge test. MAIN OUTCOME MEASURE(S) Inhibin-B concentrations on menstrual days 3 and 10. RESULT(S) Nineteen patients with normal ovarian reserve and 15 with diminished ovarian reserve had serum inhibin-B concentrations determined during ovarian reserve screening. For all patients, day 10 inhibin-B concentrations were higher than day 3. Women with normal ovarian reserve had higher inhibin-B concentrations on both days 3 and 10 than women with diminished ovarian reserve. Inhibin-B concentrations demonstrated a negative correlation with FSH levels on both cycle days 3 and 10 and a positive correlation with E2 on cycle day 10. CONCLUSION(S) Women with diminished ovarian reserve during ovarian reserve screening had reduced granulosa cell inhibin-B production compared with women with normal ovarian reserve. The lower inhibin-B concentrations may be responsible for the elevated FSH concentrations and may be indicative of the aging follicular apparatus.

Elevated level of follicular fluid vascular endothelial growth factor is a marker of diminished pregnancy potential

OBJECTIVE To evaluate whether differences in follicular fluid vascular endothelial growth factor (FF VEGF) concentrations are observed between women achieving a clinical pregnancy and those failing to conceive. DESIGN Retrospective chart review and analysis of FF VEGF concentrations. SETTING University teaching center. PATIENT(S) Fifty-seven women < or =42 years of age undergoing follicular aspiration in preparation for IVF or GIFT. INTERVENTION(S) Analysis of FF VEGF concentrations and chart review of a single IVF or GIFT cycle. MAIN OUTCOME MEASURE(S) Follicular fluid VEGF concentrations, clinical pregnancy rate, age, ampules of gonadotropins used, oocytes retrieved, peak estradiol serum concentrations, day 3 FSH levels, and fertilization rate. RESULT(S) Women who did not conceive had higher FF VEGF concentrations than women achieving a clinical pregnancy (4.409 + 2,387 versus 2.793 +/- 1,180 pg/mL: P < .001). A negative correlation was observed between FF VEGF concentrations and peak estradiol levels and number of oocytes retrieved. A positive correlation was found for FF VEGF and patients age and ampules of gonadotropins used. CONCLUSION(S) Elevated FF VEGF concentrations are associated with poor conception rates after IVF or GIFT.

Endocrine and paracrine control of oocyte development

The effects of gonadotropins on oocyte development are mediated through a variety of mechanisms, including production by granulosa cells of growth factors, cytokines, inhibins, activins, and steroids. Specific receptors for steroids, growth factors, and cytokines have been demonstrated on oocytes of several species. Gonadotropin modulation of follicular concentrations of these paracrine factors may ultimately be responsible for the precise regulation of oocyte function. As gonadotropin-releasing hormone antagonists and recombinant gonadotropins become available, the clinical use of more precise control of the endocrine, paracrine, and autocrine regulation of follicle growth and oocyte development can be more thoroughly investigated.

Neutralization of endogenous vascular endothelial growth factor depletes primordial follicles in the mouse ovary

Abstract The regulation of early follicular growth and development involves a complex interaction of autocrine, paracrine, and endocrine signals. The ability of these factors to regulate follicle growth may depend in part on the extent of vascular delivery to and perfusion of the ovary. Vascular endothelial growth factor A (VEGFA) is a major regulator of vascular physiology in the ovary. VEGFA is produced in numerous ovarian compartments and likely plays a role in the regulation of all phases of follicular growth, from preantral through preovulatory. The aim of the present study was to further evaluate the role of VEGF in early follicle growth by neutralization of endogenous VEGF or VEGF receptors. Adult mice were injected systemically and prepubertal mice were injected directly under the ovarian bursa with antibodies designed to neutralize VEGF or block interaction with its receptors in the ovary. Both systemic and intrabursal injections of VEGF antibody significantly reduced the number of primordial follicles within 1–3 days after administration without affecting primary or secondary follicle numbers. Primordial follicle numbers were not different from control levels by 30 days after VEGFA antibody administration. Administration of antibodies to the kinase domain receptor (KDR), but not the FMS-like tyrosine receptor (FLT1), for VEGF also resulted in a significant decrease in primordial follicles. These data suggest that VEGF plays a vital role in the maintenance and growth of the primordial follicle pool.

Using Second Life Virtual Simulation Environment for Mock Oral Emergency Medicine Examination

OBJECTIVES Oral examination is a method used to evaluate emergency medicine (EM) residents and is a requirement for board certification of emergency physicians. Second Life (SL) is a virtual three-dimensional (3-D) immersive learning environment that has been used for medical education. In this study we explore the use of SL virtual simulation technology to administer mock oral examinations to EM residents. METHODS This was a prospective observational study of EM residents who had previously completed mock oral examinations, participating in a similar mock oral examination case scenario conducted via SL. EM residents in this training program completed mock oral examinations in a traditional format, conducted face to face with a faculty examiner. All current residents were invited to participate in a similar case scenario conducted via SL for this study. The examinee managed the case while acting as the physician avatar and communicated via headset and microphone from a remote computer with a faculty examiner who acted as the patient avatar. Participants were surveyed regarding their experience with the traditional and virtual formats using a Likert scale. RESULTS Twenty-seven EM residents participated in the virtual oral examination. None of the examinees had used SL previously. SL proved easy for examinees to log into (92.6%) and navigate (96.3%). All felt comfortable communicating with the examiner via remote computer. Most examinees thought the SL encounter was realistic (92.6%), and many found it more realistic than the traditional format (70.3%). All examinees felt that the virtual examination was fair, objective, and conducted efficiently. A majority preferred to take oral examinations via SL over the traditional format and expressed interest in using SL for other educational experiences (66.6 and 92.6%, respectively). CONCLUSIONS Application of SL virtual simulation technology is a potential alternative to traditional mock oral examinations for EM residents.