Lifestyle factors associated with histologically derived human ovarian non-growing follicle count in reproductive age women.

STUDY QUESTION: Are lifestyle factors (smoking, BMI, alcohol use and oral contraceptive pill use) associated with the human ovarian reserve as determined by the total ovarian non-growing follicle number? SUMMARY ANSWER: Light to moderate alcohol use was significantly associated with greater ovarian non-growing follicle (NGF) count, whereas other lifestyle factors were not significantly related. WHAT IS KNOWN ALREADY: A single previous investigation has suggested that smoking and alcohol use are associated with lower ovarian follicle density. However, this investigation utilized follicle density as the outcome of interest rather than the estimated total ovarian NGF count. STUDY DESIGN, SIZE, DURATION: This cross-sectional investigation included a convenience sample of premenopausal women from two different academic sites, the University of Washington (n = 37, from 1999-2004) and the University of Oklahoma (n = 73, from 2004-2013), undergoing incidental oophorectomy at the time of hysterectomy (total n = 110, age range 21-52 years). PARTICIPANTS/MATERIALS, SETTING, METHODS: Prior to undergoing oophorectomy, participants completed detailed questionnaires regarding lifestyle exposures. Following surgery, total ovarian NGF counts were determined with systematic random sampling rules and a validated fractionator/optical dissector technique. Associations between lifestyle factors and log-transformed ovarian follicle counts were determined using multivariable linear regression. MAIN RESULTS AND THE ROLE OF CHANCE: After controlling for age, BMI, oral contraceptive pill (OCP) use, tobacco use and site of collection, cumulative alcohol use (measured in alcoholic drinks per day multiplied by years of drinking) was associated with ovarian NGF count. Women reporting light (>0 to <1 drink-years) and moderate (1-3 drink-years) alcohol use had greater NGF counts (ß = 0.75, P = 0.04, and ß = 1.00, P = 0.03; light and moderate use, respectively) as compared with non-users. Neither heavier alcohol use (>3 drink-years), BMI, OCP use, nor tobacco use were significantly associated with the ovarian NGF count. Similar patterns of association with moderate cumulative alcohol use were observed when evaluating associations with pre-antral follicles and total follicle counts. LIMITATIONS, REASONS FOR CAUTION: All participants in this convenience sample had a benign indication for hysterectomy, and therefore may not be broadly representative of the population without such an indication. Additionally, lifestyle factors were self-reported, and the sample size of the present investigation limits our ability to detect associations of smaller magnitude. WIDER IMPLICATIONS OF THE FINDINGS: While our findings are in disagreement with a single investigation that utilized human follicle density as the outcome of interest, they are consistent with many studies investigating the relationship between lifestyle factors and the age of spontaneous menopause. Furthermore, they suggest a mechanism that does not involve accelerated follicular atresia to explain the association between smoking and an earlier age of menopause. STUDY FUNDING/COMPETING INTERESTS: This investigation was funded by NIA R29-HD37360-04 (N.A.K.) and OCAST HR04-115 (K.R.H.) and by the National Institute of General Medical Sciences, Grant 1 U54GM104938 (J.D.P.). There is no conflict of interest.

Acupuncture performed before and after embryo transfer: a randomized controlled trial.

OBJECTIVE: To compare the clinical pregnancy rates in women who underwent fresh embryo transfer (ET) with and without one acupuncture session before and after the transfer using a modified Paulus protocol. STUDY DESIGN: The prospective, physician-blinded study randomized 113 women to either no intervention (n = 56) or acupuncture performed off-site (n = 57). Secondary outcomes were positive pregnancy test and live birth rates. Intent-to-treat analysis and per protocol analysis were performed. RESULTS: No difference in age and in vitro fertilization-embryo transfer (IVF-ET) parameters were detected. Compared to the control group, the treatment group had a lower pregnancy rate (43.6% vs. 64.8%, p = 0.045). More women in the control group had live births than did those in the acupuncture group (56.0% vs. 36.0%, respectively, p = 0.033). Generalized mixed models revealed that patients who received acupuncture had lower odds of clinical pregnancy than those who did not undergo acupuncture treatment (OR = 0.42, 95% CI 0.19-0.93). CONCLUSION: Our study found that acupuncture performed off-site on the day of ET was detrimental to the success of the transfer. More research is needed with a greater number of subjects to elucidate the role of acupuncture before and after ET, ideal treatment frequency, and to further explore the role of individualized acupuncture treatment on IVF-ET pregnancy rates.

A new model of reproductive aging: the decline in ovarian non-growing follicle number from birth to menopause.

BACKGROUND: The primary determinant of reproductive age in women is the number of ovarian non-growing (primordial, intermediate and primary) follicles (NGFs). To better characterize the decline in NGF number associated with aging, we have employed modern stereology techniques to determine NGF number in women from birth to menopause. METHODS: Normal human ovaries were collected from 122 women (aged 0-51 years) undergoing elective oophorectomy, organ donation or autopsy. After gross pathologic examination, systematic random sampling was utilized to obtain tissue for analysis by the fractionator/optical disector method. Models to describe the resulting decay curve were constructed and evaluated. RESULTS: NGF decay was best described by a simple power function: log (y) = ax(b) + c, where a, b and c are constants and y = NGF count at age x (R(2) = 0.84, Sums of Squares Error = 28.18 on 119 degrees of freedom). This model implies that follicles decay faster with increasing age. CONCLUSIONS: Unlike previous models of ovarian follicle depletion, our model predicts no sudden change in decay rate, but rather a constantly increasing rate. The model not only agrees well with observed ages of menopause in women, but also is more biologically plausible than previous models. Although the model represents a significant improvement compared with earlier attempts, a considerable percentage of the variation in NGF number between women cannot be explained by age alone.

Urinary beta-luteinizing hormone and beta-follicle stimulating hormone immunoenzymometric assays for population research.

OBJECTIVE: We developed assays for measurement of urinary betaLH and betaFSH under collection and storage conditions typical of non-clinical research settings. DESIGN AND METHODS: IEMAs for free betaLH and total betaFSH were validated by standard methods. Stability of urinary betaLH and betaFSH was tested across freeze-thaws and stored long term at 4 degrees C or -20 degrees C, or short term at room temperature, and with heating to dissociate the subunits. RESULTS: The IEMAs exhibited acceptable parallelism, specificity, recovery (averaging 100% for betaLH, 97% for betaFSH), imprecision (maximum within-run and between run CVs, respectively, 4.8% and 25.7% for betaLH, 5.6% and 17.0% for betaFSH), and minimum detectable dose (2.5 pmol/L for betaLH, 6.8 pmol/L for betaFSH). Urine and serum measures were highly correlated (r=0.95 for LH, 0.86 for FSH). There was no consistent decline with any storage type. Dissociation of subunits by heating was needed for betaLH, but not betaFSH. CONCLUSION: These IEMAs measure free betaLH and total betaFSH, overcoming inter-individual variability in, and collection and storage effects on, subunit dissociation, without the need for urine preservatives.

Is the short follicular phase in older women secondary to advanced or accelerated dominant follicle development?

This study sought to determine whether the shortened follicular phase in ovulatory older women is secondary to advanced (i.e. earlier) or accelerated (i.e. more rapid) folliculogenesis. Normal ovulatory women, aged 40-45 yr (n = 15) and 20-25 yr (n = 13), underwent daily venipuncture and transvaginal ultrasonography throughout the follicular phase of a spontaneous menstrual cycle (control cycle) and after pituitary down-regulation with a GnRH agonist (study cycle). As expected, the older subjects in the control cycles demonstrated an elevated d 3 FSH and a shortened follicular phase compared with the younger subjects. After release from hypothalamic-pituitary-ovarian axis suppression, the early follicular phase FSH peak occurred earlier (6.8 vs. 9.8 d; P < 0.01) and was of a greater magnitude (12.1 vs. 6.5 mIU/ml; P < 0.01) in the older subjects. The time from release of suppression until the subsequent LH surge was also shorter (17.5 vs. 20.8 d; P < 0.01) in the older group. However, the time from FSH peak to LH surge was similar in the older and younger groups (10.7 vs. 11.0 d; P = 0.74). Compared with younger women, older subjects had normal follicular phase levels of estradiol and inhibin A and lower levels of inhibin B in both control and study cycles. We conclude that the shortened follicular phase observed in older ovulatory women is due to earlier dominant follicle selection, independent of hormonal influences from the preceding luteal phase.

Age-related analysis of inhibin A, inhibin B, and activin a relative to the intercycle monotropic follicle-stimulating hormone rise in normal ovulatory women.

Previous studies have reported that the monotropic rise in FSH in older women is associated with decreased inhibin B and/or A levels and increased levels of activin A. Whereas most investigators have found decreased follicular-phase inhibin B, the roles of inhibin A and activin A as modulators of the FSH rise are unclear. The objectives of this study were to determine whether deficiencies in circulating levels of inhibin A, inhibin B, and/or activin A exist during the intercycle interval in ovulatory older (age, 40-45 yr; n = 16), compared with younger women (age, 20-25 yr; n = 13). Blood samples were obtained daily throughout one menstrual cycle and the follicular phase of the subsequent cycle and were analyzed for LH, FSH, estradiol, inhibin A and B, and activin A. Despite significant FSH elevation, no deficiencies in inhibin A, activin A, or estradiol were detected in older subjects. In fact, inhibin A was significantly higher in older participants during the intercycle phase (P = 0.01), whereas inhibin B was significantly lower. Thus, the monotropic rise in FSH does not appear to result from changes in inhibin A or activin A, supporting the concept that inhibin B plays a critical role in mediating the FSH rise in older women.

Antral follicle count and serum follicle-stimulating hormone levels to assess functional ovarian age.

OBJECTIVE: The objective of this study was 2-fold. The first was to estimate side-to-side variation in antral follicle counts. The second was to determine whether basal follicle-stimulating hormone levels on days 2, 3, and 4 of the same menstrual cycle are significantly different. METHODS: Forty-one patients between the ages of 20 and 42 years undergoing monitoring for in vitro fertilization-embryo transfer were evaluated ultrasonographically for antral follicle number. The antral follicle counts were determined for each ovary by experienced ultrasonographers at the time of suppression check ultrasonography. In a separate study, 62 normal subjects (ages 20-25 and 40-45 years) underwent serial sequential serum follicle-stimulating hormone determinations on days 2-4 of the menstrual cycle, and these levels were compared. RESULTS: There was no significant difference between right and left antral follicle counts (P =.30). Serial follicle-stimulating hormone values were not significantly different on days 2, 3, or 4 of the menstrual cycle (P =.22). CONCLUSION: There is no significant difference between right-sided and left-sided antral follicle counts within the same individual. In turn, there is no significant difference in serial follicle-stimulating hormone levels on days 2, 3, or 4 of the menstrual cycle. LEVEL OF EVIDENCE: III

Comparison of basal follicle-stimulating hormone versus the clomiphene citrate challenge test for ovarian reserve screening.

OBJECTIVE: To compare the value of basal follicle-stimulating hormone (FSH) measurement vs. the clomiphene citrate challenge test (CCCT) in predicting the ability to achieve a pregnancy in women who are undergoing infertility treatment. DESIGN: Meta-analysis. SETTING: All studies that evaluated either basal FSH or the CCCT for determining the likelihood of pregnancy. PATIENT(S): Infertility population undergoing treatment, which was defined as patients undergoing ovulation induction, IUI, or in vitro fertilization (IVF). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Diagnostic test characteristics were calculated and pooled using standard methods. Inability to achieve a pregnancy with treatment was considered as the "disease." RESULT(S): Twelve studies on basal FSH (with 6296 patients, mean age 33.8) and seven studies on the CCCT (with 1352 patients, mean age 34.5) fit our criteria and were analyzed. For basal FSH and the CCCT, the sensitivities were 6.6% (95% confidence interval [CI] 5.9, 7.3%) and 25.9% (95% CI 23.0, 29.0%), respectively, and specificities were 99.6% (95% CI 99.1, 99.9%) and 98.1% (95% CI 96.5, 99.1%), respectively. For "disease" prevalence ranging from 40%-100%, for basal FSH and the CCCT, the positive predictive values ranged from 91.7%-100% and 90.1%-100%, respectively, and negative predictive values ranged from 61.5%-0.0% and 66.5%-0.0%, respectively. CONCLUSION(S): Basal FSH and the CCCT are similar in predicting the ability to achieve a clinical pregnancy in women undergoing infertility treatment. With either test, a normal result is not useful, but an abnormal result virtually confirms that pregnancy will not occur with treatment.

Intraoperative ultrasound during a laparoscopic myomectomy.

OBJECTIVE: To report a gynecologic use of a laparoscopic ultrasound transducer to isolate a myoma for surgical removal. DESIGN: Case report. SETTING: University-based infertility practice. PATIENT(S): A 44-year-old woman gravida 1 para 1 with history of a first trimester miscarriage who desired pregnancy as a participant in the donor egg program. INTERVENTION(S): Before she entered the assisted reproduction program, a patient was found to have a myoma that was greater than 2 cm with both intramural and submucosal components. During the laparoscopic evaluation, a laparoscopic ultrasound transducer helped identify and properly locate the myoma in what otherwise appeared to be a normal uterus. Appropriate laparoscopic hysterotomy incision was then made, thereby minimizing uterine trauma. MAIN OUTCOME MEASURE(S): Appropriately placed hysterotomy incision and successful reconstruction of uterus. RESULT(S): After the successful laparoscopic myomectomy, the patient achieved a pregnancy in our donor oocyte program. CONCLUSION(S): Laparoscopic intraoperative ultrasound can help gynecologic surgeons complete a laparoscopic myomectomy.

Follicular development, ovulation, and corpus luteum formation in cryopreserved human ovarian tissue after xenotransplantation.

OBJECTIVE: To assess the competency of human frozen/thawed ovarian follicles matured in xenografts to form functioning corpora luteae after human chorionic gonadotropin (hCG) administration. DESIGN: Prospective controlled animal study. SETTING: University research laboratory. PATIENT(S): Three women (19, 28, and 36 years) who underwent oophorectomy. ANIMAL(S): Nineteen female severe combined immunodeficient (SCID) mice. INTERVENTION(S): Cryopreserved human ovarian tissues were grafted into the s.c. space of bilaterally oophorectomized SCID mice. All the animals were stimulated with pregnant mare's serum gonadotropin (PMSG) for 4 weeks starting from 16 weeks after transplantation. Twelve animals were injected with hCG at the end of gonadotropin stimulation. MAIN OUTCOME MEASURE(S): [1] The rate of grafts with growing follicles, with antral follicles, and/or with corpora luteae. [2] The histologic assessment of follicles and corpora luteae. [3] The serum progesterone and estradiol level in animals with corpus luteum in the grafts. RESULT(S): [1] The rate of grafts with growing follicles and with corpora luteae was 33% to 100%, and 28% to 50%, respectively. [2] Corpora luteae in xenografts were all morphologically normal. [3] The progesterone levels were all above 3.0 ng/mL. CONCLUSION(S): This study showed that the cryopreserved human ovarian follicles can be matured to a stage at which they can form functioning corpora luteae in the host animal.

Factors influencing pregnancy rates with a combined clomiphene citrate/gonadotropin protocol for non-assisted reproductive technology fertility treatment.

OBJECTIVE: To determine the effectiveness of a combined clomiphene citrate/gonadotropin protocol in a general infertility population and to evaluate factors influencing pregnancy rates obtained with this protocol. DESIGN: A retrospective chart review. SETTING; University-based infertility clinic. PATIENT(S): Two hundred forty-eight patients undergoing 658 cycles of minimal stimulation (MS) protocol from 1996-2000. INTERVENTION(S): Patients underwent treatment with clomiphene citrate and gonadotropin, often followed by intrauterine insemination. MAIN OUTCOME MEASURE(S): Clinical pregnancy rates. RESULT(S): Overall, the clinical pregnancy rate was 7.1% per cycle (n = 248 patients and 658 cycles). The age range of the patients was 24-47 years (mean +/- SD = 36.5 +/- 4.9) with 8.7% noted to have ovulatory dysfunction. Pregnancy rates varied significantly (P<.05) with patient age (9.3% in women <40 years vs. 2.4% in women > or =40), duration of infertility (9.0% in women with < or =3 years of infertility vs. 2.2% in women with >3 years of infertility) and number of follicles produced during stimulation (9.1% in women with > or =3 follicles vs. 4.6% in women with <3 follicles). CONCLUSION(S): The effectiveness of the MS protocol in a general infertility population with a predominantly ovulatory status is much less than that previously reported in a younger patient population with a significant rate of ovulatory dysfunction. This protocol does not appear to lead to pregnancy rates higher than that reported for clomiphene citrate/intrauterine insemination (IUI) cycles. The clinical pregnancy rates using a minimal stimulation protocol are particularly compromised in women over 40, those with a longer duration of infertility or those who produce few follicles during stimulation.

Reproductive aging and variability in the ovarian antral follicle count: application in the clinical setting.

OBJECTIVE: To determine the extent of intercycle and interobserver variability in antral follicle (AF) count and their impact on stimulation quality in IVF. DESIGN: Prospective evaluation of the impact on AF count of GnRH agonist down-regulation and interobserver variability. Retrospective evaluation of intercycle variability in AF count. SETTING: University ART clinic. PATIENT(S): Twenty subjects were used to evaluate the effect of GnRH agonist down-regulation upon AF count; six of whom were used to evaluate interobserver variability. Fifty patients experiencing two or three cycles of IVF within a 1-year interval. INTERVENTION(S): Transvaginal ultrasound exams before and after down-regulation with a GnRH agonist. Videotaped day-3 transvaginal ultrasound exams. MAIN OUTCOME MEASURE(S): [1] Intercycle and interobserver variability in antral follicle count. [2] Oocytes retrieved, peak estradiol, gonadotropin dose, duration of stimulation and cancellation rates. RESULT(S): There is moderate intercycle and interobserver variability in AF counts. GnRH agonist down-regulation does not significantly change AF count. In infertility patients undergoing IVF, paired analysis between the low- and high-AF count cycles did not show a difference in quality of stimulation or cycle cancellation rates. CONCLUSIONS: Within an individual patient, higher AF count in a given cycle was not predictive of better stimulation compared with the case of a lower count cycle.

The epidemiology of female rape victims who seek immediate medical care: temporal trends in the incidence of sexual assault and acquaintance rape.

Women who seek medical care following sexual assault are usually evaluated and treated in an emergency department (ED). Therefore, EDs can be an important source of sexual assault surveillance data. The authors compared the incidence of sexual assault presenting for emergency care in a single county during July to November of 1974 and 1991. Participants included all female sexual assault victims aged 14 and older who presented for ED evaluation. Treating physicians prospectively collected data using standardized forms. The z statistic was used to compare sexual assault incidence. There was a 60% increase in the incidence of sexual assault victims presenting for emergency care in 1991 compared to 1974, primarily due to an increase in the incidence of women presenting to the ED after rapes by known assailants. In contrast, the annual incidence of reported stranger assaults was similar in the two study years.

Ovarian primordial and nongrowing follicle counts according to the Stages of Reproductive Aging Workshop (STRAW) staging system.

OBJECTIVE: The aim of this study was to characterize the ovarian primordial and nongrowing follicle number according to the Stages of Reproductive Aging Workshop (STRAW) staging system as defined by menstrual cycle characteristics. METHODS: Normal ovaries were collected from 63 women (age 26-52 y) undergoing oophorectomy for benign indications. Before surgical operation, each participant completed a detailed questionnaire collecting information regarding menstrual cycle characteristics and was classified by bleeding patterns into STRAW stages -4, -3, -2, and -1. A single ovary was selected for the determination of ovarian primordial and total nongrowing follicle number using a validated fractionator/optical disector method. A subset of the participants (n = 43) underwent transvaginal ultrasound examination for the determination of the ovarian antral follicle count and serum measurements of follicle-stimulating hormone, estradiol, antimüllerian hormone, and inhibin B. All measurements were obtained within 2 weeks of surgical operation, irrespective of cycle day. RESULTS: Significant differences were identified in ovarian primordial (P < 0.0001) and nongrowing follicle (P < 0.0001) counts across the STRAW stages. In post hoc testing, the differences in primordial follicle counts were significant between each of the STRAW stages. Significant differences were also identified in serum levels of antimüllerian hormone, follicle-stimulating hormone, and ovarian antral follicle count across the STRAW stages. CONCLUSIONS: Progression through the STRAW stages as defined by menstrual cycle characteristics is associated with progressive and significant decreases in the ovarian primordial follicle number.

Factors affecting success rates in two concurrent clinical IVF trials: an examination of potential explanations for the difference in pregnancy rates between the United States and Europe.

OBJECTIVE: To compare a US clinical trial of gonadotropin therapy for IVF with a similar European trial to determine what factors may explain the higher clinical pregnancy rate in the US trial. DESIGN: Comparison of baseline, treatment, and outcome variables in the United States (US) and European trials. SETTING: IVF practices in the US (n=4) and Europe (n=6). PATIENT(S): 297 women undergoing IVF. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate. RESULT(S): Clinical pregnancy rates were 43.4% in the US compared with 29.7% in Europe (p=0.016), with a live birth rate of 38.2% versus 27.6% (p=0.064). This difference in clinical pregnancy rate could not be explained by differences in the US versus Europe for number of embryos transferred (2.3 vs. 2.6) or female age (34.6 vs. 30.4). Although the starting dose of gonadotropin was higher in the US trial compared with the European trial (300 versus 225 IU), the total dose of gonadotropin was only slightly higher in the US. In multiple logistic regression analysis of 81 pretransfer variables on clinical pregnancy, the only two found to be significant predictors of outcome were baseline endometrial thickness following down-regulation and number of days of gonadotropin treatment. CONCLUSION(S): This study suggests the possibility that US pregnancy rates may be higher in part because of differences in down-regulation or gonadotropin dosing. Other factors not assessed in these studies or in national datasets likely also contribute to the difference in pregnancy rates.

Clinical efficacy of highly purified urinary FSH versus recombinant FSH in volunteers undergoing controlled ovarian stimulation for in vitro fertilization: a randomized, multicenter, investigator-blind trial.

OBJECTIVE: To compare the efficacy of highly purified human urinary follicle stimulating hormone (HP-hFSH) versus human recombinant follitropin-alpha (rFSH) in volunteers undergoing controlled ovarian stimulation for IVF. DESIGN: A randomized, controlled, investigator-blind trial. SETTING: Four assisted reproductive technology centers. PATIENT(S): One hundred fifty-two IVF patients. INTERVENTION(S): Volunteers, aged 18-39, were randomized to HP-hFSH (n = 76) versus rFSH (n = 76) at a starting dose of 300 IU in down-regulated cycles. MAIN OUTCOME MEASURE(S): Number of oocytes, clinical pregnancy rate, and live birth rate with HP-hFSH versus rFSH. RESULT(S): The total IU of gonadotropin used did not differ between the two groups. There was no difference in number of oocytes retrieved with HP-hFSH (mean = 16.3) compared with rFSH (mean = 17.1), confidence interval (CI) of difference = -3.79 to +2.18. Clinical pregnancy rate, as defined by the presence of a gestational sac, was 48.7% (CI = 37.0%-60.4%) with HP-hFSH versus 44.7% (CI = 33.3%-56.6%) with rFSH (CI of difference = -11.9% to +19.8%). Live birth rate was 38.2% (29 of 76) in both groups (CI = 27.2%-50.0%), for a difference between groups of 0.0% (CI of the difference = -15.4% to +15.4%). CONCLUSION(S): There were no statistically significant differences in mean oocyte number, clinical pregnancy rate, or live birth rate between HP-hFSH versus rFSH.

Estimating human ovarian non-growing follicle number: the application of modern stereology techniques to an old problem.

BACKGROUND Previous published reports on the number of non-growing follicles (NGFs) in the human ovary have employed model-based methods for number estimates. These methods are time-intensive, and require correction factors and assumptions that ultimately limit their accuracy. Here, we describe the modification, application and validation of a modern fractionator/optical disector technique for the estimation of human ovarian NGF number. METHODS Forty-eight pairs of normal human ovaries were collected from women (age 8-51 years) undergoing elective bilateral oophorectomy, organ donation, or from autopsy. After gross pathologic examination, systematic random sampling was utilized to obtain tissue for analysis by the fractionator/optical disector method. The precision of individual NGF counts was determined by calculating the observed coefficient of error (OCE). Intra-observer variability and variation in NGF number between ovaries within a pair were also determined. RESULTS The mean OCE was 16.6% with larger variations observed at lower follicle counts. In recount experiments of the same ovary, NGF number estimates varied by 15-29%, except at very low follicle counts where variation was greater, but absolute differences were small. There was no significant difference in NGF number between ovaries within a pair (Wilcoxon signed rank test, P = 0.81). CONCLUSIONS Modern stereology methods provide an unbiased, efficient method for estimating NGF number in the human ovary. Both ovaries within a pair contain similar numbers of NGFs.