Richard J. Hackett

Age-related normograms of serum antimüllerian hormone levels in a population of infertile women: a multicenter study

OBJECTIVE To produce age-related normograms for serum antimüllerian hormone (AMH) level in infertile women without polycystic ovaries (non-PCO). DESIGN Retrospective cohort analysis. SETTING Fifteen academic reproductive centers. PATIENT(S) A total of 3,871 infertile women. INTERVENTION(S) Blood sampling for AMH level. MAIN OUTCOME MEASURE(S) Serum AMH levels and correlation between age and different percentiles of AMH. RESULT(S) Age-related normograms for the 3rd, 10th, 25th, 50th, 75th, 90th, and 97th percentiles of AMH were produced. We found that the curves of AMH by age for the 3rd to 50th percentiles fit the model and appearance of linear relation, whereas the curves of >75th percentiles fit cubic relation. There were significant differences in AMH and FSH levels and in antral follicle count (AFC) among women aged 24-33 years, 34-38 years, and ≥39 years. Multivariate stepwise linear regression analysis of FSH, age, AFC, and the type of AMH kit as predictors of AMH level shows that all variables are independently associated with AMH level, in the following order: AFC, FSH, type of AMH kit, and age. CONCLUSION(S) Age-related normograms in non-PCO infertile women for the 3rd to 97th percentiles were produced. These normograms could provide a reference guide for the clinician to consult women with infertility. However, future validation with longitudinal data is still needed.

Serum triple-marker screening in in vitro fertilization and naturally conceived pregnancies

Objective To determine whether results of second-trimester maternal serum triple-marker screening for Down syndrome and open neural tube defects in singleton pregnancies conceived from in vitro fertilization (IVF) differ from those of pregnancies conceived spontaneously. Methods The screen-positive rates and triple-marker levels of patients conceiving singleton pregnancies by IVF were compared to age-adjusted standards. Results Sixty-nine singleton IVF pregnancies with maternal serum screening were identified. Twenty-one (30.4%) of the 69 IVF singleton pregnancies had a positive screen for Down syndrome compared with a 14.4% expected screen-positive rate for the maternal age distribution in our observed sample (P = .013). The screen-positive rate for open neural tube defects in the measured population was similar to anticipated values based on historic controls (5.8% in IVF patients versus 5.3% in the total population). The median levels of the triple markers were 0.95 multiples of the median (MoM) for alpha-fetoprotein (AFP), 0.90 MoM for unconjugated estriol (E3), and 1.22 MoM for hCG. Conclusion The increased hCG levels as well as the slightly lower AFP and unconjugated E3 levels may contribute to the higher Down syndrome screen-positive rate in this IVF singleton population. These results may be due to the number of embryos transferred, the maternal hormonal environment of the IVF process, or other factors. Pregnancies conceived by IVF may be twice as likely to have a positive maternal serum screening test. As additional data are collected, corrected standards should be determined.

Rigorous thermal control during intracytoplasmic sperm injection stabilizes the meiotic spindle and improves fertilization and pregnancy rates

OBJECTIVE To examine the effects of different thermodynamic control systems on the temperature stability of human eggs during in vitro manipulation, with the integrity of meiotic spindles imaged using the LC-PolScope (Cambridge Research & Instrumentation, Inc., Woburn, MA). DESIGN We performed intracytoplasmic sperm injection (ICSI) and/or imaging of eggs with the temperature regulated by three different systems: thermostated coverslip (system 1), thermostated coverslip combined with objective heater (system 2), and conventional stage warmer (system 3). SETTING Academic in vitro fertilization clinic. PATIENT(S) Oocytes were aspirated from stimulated ovaries of patients undergoing oocyte retrieval for ICSI. INTERVENTION(S) Measurement of temperature regulation in media surrounding eggs during in vitro manipulation and imaging. MAIN OUTCOME MEASURE(S) Rate of oocytes with spindles, fertilization rates, and clinical pregnancy rates after ICSI. RESULT(S) We imaged spindles in more oocytes with system 2 (81.2%) than with system 1 (61.4%). Spindles could not be imaged for system 3 because of technical limitations. Fertilization rates were significantly higher when oocytes were imaged and used for ICSI with system 2 (78.8%) than with system 1 (56.7%) or system 3 (64.0%). Most importantly, a significantly higher clinical pregnancy rate was observed when oocytes were manipulated with system 2 (51.7%) than with system 1 (25.0%) or system 3 (23.1%). No differences were found in average ages, number of previous cycles, number of eggs, or day 3 FSH or E2 levels among groups. CONCLUSION(S) Imaging meiotic spindles with the PolScope provides an intracellular thermostat during ICSI. Rigorous thermal control during ICSI stabilized spindles and increased the fertilization and clinical pregnancy rates achieved after ICSI. The presence of birefringent spindles in living human eggs served as a monitor for in vitro conditions.

Steroid sulphatase activity in the human ovarian corpus luteum, stroma, and follicle : comparison to activity in other tissues and the placenta

Steroid sulfatase activity was measured in 89 human samples, using dehydroepiandrosterone sulfate (DHEAS) as substrate. The lowest activity was that of follicular fluid which was significantly lower than that of other tissues tested (each P less than 0.01). The steroid sulfatase activity of ovarian tissue taken collectively (corpus luteum, stroma, and follicles) was higher than that of other tissues taken collectively (abdominal skin, uterus, and fallopian tube) (P less than 0.001), and the steroid sulfatase activity of either the follicle (P less than 0.01) or the stroma (P less than 0.05) was significantly greater than that of the corpus luteum. The geometric mean steroid sulfatase activity of the placenta was significantly higher than other tissues tested (each P less than 0.01) and was 22-fold higher than that of the follicle, the tissue with the next highest activity. These data indicate that the human ovary (particularly the stroma and follicle) is capable of utilizing DHEAS, an adrenal product, as a substrate for production of other androgens such as dehydroepiandrosterone (DHEA), androstenedione, and testosterone.

Use of in-cycle antimüllerian hormone levels to predict cycle outcome

OBJECTIVE The goal of this work is to expand the usefulness of antimüllerian hormone (AMH) in predicting in vitro fertilization cycle outcome by demonstrating that AMH concentration obtained in an ongoing treatment cycle predicts both oocyte number and pregnancy. STUDY DESIGN Serum samples were obtained from 190 in vitro fertilization patients at onset of follicle-stimulating hormone stimulation. These were analyzed retrospectively during a single cycle in which clinicians were blinded to the results. Our major outcome measures were the number of oocytes obtained and ongoing pregnancy. RESULTS Patients with an initial AMH concentration of >3 ng/mL were found to produce a mean of 19.8 oocytes and had an ongoing pregnancy rate of 60.3%. In contrast, those with AMH values of ≤1 ng/mL yielded a mean of 6.2 oocytes and had an ongoing pregnancy rate of 23.4% (P < .0001 for both). CONCLUSION Greater AMH serum concentration strongly predicts an increased number of oocytes and ongoing pregnancy (P ≤ .0001).

5α-reductase 1 and 2 expression and activity in human ovarian follicles, stroma and corpus luteum as compared to neonatal foreskin

5alpha-Reductase is the steroidogenic enzyme which reduces testosterone to 5alpha-dihydrotestosterone. In the human two different enzymes have been described, 5alpha-reductase 1 and 2. The present investigations were undertaken to determine whether 5alpha-reductase 1 and 2 were expressed in the human ovary, and to determine the relative activity of the two enzymes in various ovarian tissues. The ovary apparently expressed mRNA for only 5alpha-reductase 1, whereas the foreskin expressed both 5alpha-reductase 1 and 2. We compared the 5alpha-reductase activity at both pH 5.5 (optimum for 5alpha-reductase 2 activity) and 8.0 (optimum for 5alpha-reductase 1 activity). 5alpha-reductase activity of foreskin at pH 5.5 was 3900 times higher than small follicles, 1500 times higher than ovarian stroma, and 240 times higher than corpora lutea (all P < 0.01). 5alpha-reductase activity of corpora lutea at pH 5.5 was 17-fold higher than that of follicles (P < 0.01) and 6.5-fold higher than that of ovarian stroma (P < 0.05). 5alpha-Reductase activity of foreskin at pH 8.0 was 93 times higher than small follicles, 51 times higher than corpora lutea, and 170 times higher than ovarian stroma (all P < 0.01). The ratio of 5alpha-reductase activity at pH 5.5 to that at pH 8.0 was higher in foreskin than in corpus luteum (P < 0.05), ovarian stroma (P < 0.01), or ovarian follicles (P < 0.01). The ratio was lower in ovarian follicles than in stroma or corpus luteum (both P < 0.05).

Steroid sulfatase in the human ovary and placenta: enzyme kinetics and phosphate inhibition.

In 5 placental homogenates the Km of steroid sulfatase for DHEA sulfate increased from 15.4 in Tris buffer to 26.8 microM in phosphate (both buffers 0.1 M, pH 7.4), P less than 0.05. In 3 pooled ovarian preparations the Km increased from 14.3 microM in Tris to 33.0 microM in phosphate, P less than 0.01. There was no significant difference between the ovarian and placental values for Km in either Tris or phosphate (P greater than 0.5), and the increase in the Km produced by phosphate in ovarian tissue was not significantly different from that in the placenta (P greater than 0.5). In the placentas the Vmax in Tris was 1420 pmol/min/mg protein and this fell to 523 pmol/min/mg protein in phosphate (P less than 0.005). The Vmax was 50-fold higher in the placenta than in the ovary in either Tris or phosphate (both P less than 0.001). In the ovary, the Vmax was 27.6 pmol/min/mg protein in Tris and 11.0 pmol/min/mg protein in phosphate (P less than 0.05). The reduction of Vmax produced by phosphate in the ovary was not significantly different from that in the placenta (P greater than 0.5). The slope of the 1/v vs 1/S plot (Km/Vmax) increased 4.7-fold in the placentas and 5.8-fold in the ovaries in phosphate over that in Tris (both P less than 0.001); the increase in the placentas was not significantly different from that in the ovaries (P greater than 0.5). Phosphate ion acts as a mixed inhibitor of both placental and ovarian steroid sulfatase.

Effects of fetal sex, stage of gestation, dibutyryl cyclic adenosine monophosphate, and gonadotropin releasing hormone on secretion of human chorionic gonadotropin by placental explants in vitro

Explants from 16 term and 6 midtrimester placentas were cultured for 6 days. Statistically significant increases in secretion of human chorionic gonadotropin occurred in control medium cultures of both term and midtrimester explants during the 6-day culture period (p less than 0.01). Statistically significant increases in secretion of human chorionic gonadotropin were produced by 2 mmol/L dibutyryl cyclic adenosine monophosphate in both the term (p less than 0.01) and the midtrimester (p less than 0.01) explants. There was no effect of gonadotropin releasing hormone. The ratio of human chorionic gonadotropin secretion from midtrimester explants to that from term explants varied under different conditions, dropping from twentyfold in day 1 cultures to elevenfold for maximum secretion produced after culture in control medium for up to 6 days. A further drop in the ratio to fourfold was observed for the maximal response to 2 mmol/L dibutyryl cyclic adenosine monophosphate treatment. Explants from term female infants produced significantly more human chorionic gonadotropin than those from term male infants (p less than 0.05), but the sex difference disappeared after stimulation with 2 mmol/L dibutyryl cyclic adenosine monophosphate.

Testosterone, a Follicular Regulator: Key to Anovulation

To study the interrelationships of steroids within the follicle, combined 6-h infusions of [3H]dehydroepiandrosterone sulfate and [14C] testosterone ([14C]T) were performed in four normal women treated with menotropins who were undergoing medically indicated surgery. The concentrations of tracer and/or nonisotopic dehydroepiandrosterone sulfate, androst-5-ene-3 beta,17 beta-diol sulfate, androst-5-ene-3 beta,17 beta-diol, dehydroepiandrosterone, androstenedione, T, dihydrotestosterone, estrone (E1), and estradiol (E2) were determined in arterial and venous blood and follicular fluid. The log-transformed product/precursor ratio of [3H]dihydrotestosterone/[3H]T in follicular fluid was negatively correlated with the log-transformed follicular concentrations of E1 (P = 0.01) and E2 (P = 0.02), suggesting a reciprocal relationship between 5 alpha-reductase and follicular E1 and E2. E2 and T were positively correlated in follicular fluid (r = 0.84; P = 0.0003), suggesting a stimulatory action of follicular T on aromatase. These findings along with extensive published data suggest that follicular T functions as a follicular regulator, enhancing follicular aromatase activity when adequate amounts of FSH are available. These conclusions have important implications with regard to mechanisms for selecting the dominant follicle and producing atresia in the remaining cohort of follicles, and they describe a final common path in the pathophysiology of anovulation.