Nancy A. Klein

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.

Failure of oocyte activation after intracytoplasmic sperm injection using round-headed sperm.

OBJECTIVE To examine the outcome of intracytoplasmic sperm injection (ICSI) with round-headed sperm (globozoospermia). DESIGN Retrospective analysis. SETTING In vitro fertilization laboratory with extensive ICSI experience. PATIENT(S) A patient couple with infertility because of globozoospermia seeking ICSI treatment. MAIN OUTCOME MEASURE(S) Fertilization, cleavage, and pregnancy rates. INTERVENTION(S) Intracytoplasmic sperm injection and calcium ionophore. RESULT(S) This couple experienced only 7% fertilization after ICSI in their first cycle. Treatment of the unfertilized oocytes with calcium ionophore 20 hours after ICSI-induced fertilization and cleavage of 70% of the oocytes. Embryo quality was fair to good. On the second cycle, 8 of the injected oocytes were treated with ionophore immediately after ICSI and the remaining 20 oocytes were untreated. Normal fertilization was achieved in 75% of the treated and 10% of the untreated oocytes. Treatment of these unfertilized oocytes with ionophore 20 hours after ICSI resulted in fertilization in 73%. Pregnancy was not achieved after either ICSI cycle. Ultrastructural analysis indicated multiple structural abnormalities in the sperm. CONCLUSION(S) These results indicate that the round-headed sperm from this patient were incapable of oocyte activation after ICSI. This may be the reason for the frequent ICSI fertilization failure seen with this condition. Current ICSI procedures may not always overcome the infertility associated with globozoospermia, and further study of the etiology of this condition is needed.

Urinary Estrone Conjugate and Pregnanediol 3-Glucuronide Enzyme Immunoassays for Population Research

BACKGROUND Monitoring of reproductive steroid hormones at the population level requires frequent measurements, hormones or metabolites that remain stable under less than ideal collection and storage conditions, a long-term supply of antibodies, and assays useful for a range of populations. We developed enzyme immunoassays for urinary pregnanediol 3-glucuronide (PDG) and estrone conjugates (E1Cs) that meet these criteria. METHODS Enzyme immunoassays based on monoclonal antibodies were evaluated for specificity, detection limit, parallelism, recovery, and imprecision. Paired urine and serum specimens were analyzed throughout menstrual cycles of 30 US women. Assay application in different populations was examined with 23 US and 42 Bangladeshi specimens. Metabolite stability in urine was evaluated for 0-8 days at room temperature and for 0-10 freeze-thaw cycles. RESULTS Recoveries were 108% for the PDG assay and 105% for the E1C assay. Serially diluted specimens exhibited parallelism with calibration curves in both assays. Inter- and intraassay CVs were <11%. Urinary and serum concentrations were highly correlated: r = 0.93 for E1C-estradiol; r = 0.98 for PDG-progesterone. All Bangladeshi and US specimens were above detection limits (PDG, 21 nmol/L; E1C, 0.27 nmol/L). Bangladeshi women had lower follicular phase PDG and lower luteal phase PDG and E1Cs than US women. Stability experiments showed a maximum decrease in concentration for each metabolite of <4% per day at room temperature and no significant decrease associated with number of freeze-thaw cycles. CONCLUSIONS These enzyme immunoassays can be used for the field conditions and population variation in hormone metabolite concentrations encountered in cross-cultural research.

Bacteria in the transfer catheter tip influence the live-birth rate after in vitro fertilization

OBJECTIVE To assess the impact of individual bacteria isolated from the vagina and tip of the embryo transfer catheter on live-birth rates. DESIGN Prospective clinical study. SETTING Infertility outpatient clinic of a university hospital. PATIENT(S) Ninety-one women undergoing IVF-ET. INTERVENTION(S) Cultures were obtained from the vagina for aerobic and anaerobic bacteria at the time of both sonographic egg retrieval and embryo transfer and from the tip of the embryo transfer catheter. Doxycycline treatment was started after egg retrieval. MAIN OUTCOME MEASURE(S) The live birth of one or more neonates. RESULT(S) Doxycycline had no substantial impact on the recovery of individual vaginal bacteria or on bacterial vaginosis. An increase in live-birth rate was associated with the recovery of hydrogen peroxide-producing Lactobacillus from the vagina (P=0.01) and from the embryo transfer catheter (P=0.01). In contrast, a reduction in live-birth rate was associated with recovery of Streptococcus viridans (S. viridans) from the embryo transfer catheter tip (P=0.04). CONCLUSION(S) In the setting of IVF-ET, prophylactic doxycycline had little effect on vaginal bacteria. Specific bacteria recovered from the embryo transfer catheter appear associated with a detrimental or beneficial effect or with no effect on live-birth rates.

Endocrine changes of the perimenopause.

Reproductive aging in women is closely tied to the loss of ovarian follicles through atresia. The sentinel endocrinologic finding is the monotropic FSH rise, associated with a decline in ovarian inhibin B secretion. Fertility becomes significantly compromised long before overt clinical signs occur, such as cycle irregularity. Compromised fertility is primarily related to oocyte dysfunction. As women with regular cycles near the end of the reproductive years, the following changes are usually manifested: 1) the selection and development of a dominant follicle occurs earlier; 2) there is earlier ovulation; 3) there is a short follicular phase and total cycle length; and 4) ovarian steroid secretion is normal. The relationships, if any, between the monotropic FSH rise, accelerated follicular atresia, shortened follicular phase, and oocyte quality remain to be determined. The next phase of reproductive aging is the perimenopause. Lack of predictability is the rule with regard to the nature and duration of the perimenopause. Long cycles are interspersed with short ones, and intermittent ovulatory cycles are intermingled with periods that are hormonally indistinct from the postmenopausal state. Even after the last menstrual period, evidence of intermittent ovarian estradiol production may still be detected. Although fertility is severely compromised during the perimenopause, ovulation may occur without warning and contraception must be practiced if pregnancy is not desired. Further studies are needed to elucidate the factors contributing to oocyte abnormalities in women of advanced reproductive age, as well as the factors that determine the rate of follicle atresia and the length of the reproductive life span.

Inhibin and reproductive aging in women

The monotropic FSH rise is the sentinel endocrine event that first indicates a woman is approaching the end of her reproductive potential. While a deficiency in inhibin has long been postulated as the immediate cause of the monotropic FSH rise, this has only recently been demonstrated to actually occur. It is our current hypothesis that when the number of preantral follicles in both ovaries drop below a threshold, then there is a subtle decrease in inhibin B which leads to the monotropic FSH rise which, in turn, accelerates follicular depletion and the attainment of the menopause.

The Gonadotropin Secretion Pattern in Normal Women of Advanced Reproductive Age in Relation to the Monotropic FSH Rise

Women of advanced reproductive age are known to demonstrate subtle FSH elevations (monotropic FSH rise) while still retaining ovulatory function. Te purpose of this study was to investigate the hypothesis that the physiologic basis for the monotropic FSH rise is an alteration in the secretion pattern of the GnRH pulse generator. The subjects were 11 normal women age 40–45 years who underwent 24 hours of frequent blood sampling in the follicular (EF) and/or midluteal (ML) phases of spontaneous menstrual cycles. The controls were 11 normal women age 20–25 years. Tlie respective gonadotropin secretion patterns were analyzed for LH pulse frequency, mean LH and FSH levels, and LH pulse amplitude. Time were no differences between the groups for estradiol (E2) and progesterone when the respective cycle phases were compared. The 24-hour mean FSH level was significantly increased in the older women in both the EF and ML phases. There were no differences between the groups in either cycle phase for LH pulse frequency, LH pulse amplitude, and mean LH levels. The results lend no support to the hypothesis that a slowing or other alteration of the GnRH pulse generator is the basis for the monotropic FSH rise in older ovulatory women. Other possibilities include the dynamics of E2 secretion or changes in FSH-modulating peptides (ie, inhibin) in these women.

The pattern of infertility diagnoses in women of advanced reproductive age

OBJECTIVE Our intention was to determine whether there is a unique pattern of infertility diagnoses in older infertile couples. STUDY DESIGN The design of this study was a retrospective chart review study. It was performed in a tertiary referral reproductive medicine unit. There were 2 groups of patients-couples: group 1, female partner aged 20-29 (n = 105) at presentation; group 2, female partner aged 40-45 (n = 112) at presentation. All women underwent infertility evaluations between 1989 and 1994. There were no interventions. The prevalence of standard infertility diagnoses was the main outcome measure. RESULTS The prevalence of 8 major infertility diagnoses in the younger and older groups (each couple could have >/=1 diagnosis) was as follows: (1) ovulatory factor-younger group, 56%; older group, 30%; (2) tubal factor-younger group, 34%; older group, 29%; (3) endometriosis-younger group, 13%; older group, 17%; (4) uterine factor-younger group, 1%; older group, 5%; (5) cervical factor-younger group, 4%; older group, 1%; (6) luteal deficiency-younger group, 4%; older group, 10%; (7) male factor-younger group, 32%; older group, 45%; (8) unexplained-younger group, 5%; older group, 10%. The only significant difference was an increase in ovulatory factor in the younger group. CONCLUSIONS There is no unique pattern of infertility diagnoses in women of advanced reproductive age as seen at a tertiary referral center. We speculate that a high false-positive rate associated with standard infertility tests and a different referral pattern for older couples obscures any real differences in the etiology of infertility in older couples.

Statistical analysis of factors affecting fertilization rates and clinical outcome associated with intracytoplasmic sperm injection

OBJECTIVE To identify and evaluate the statistically significant predictors of intracytoplasmic sperm injection (ICSI) fertilization rates and clinical pregnancy in a single population using appropriate statistical techniques. DESIGN Retrospective study. SETTING Fertility and Endocrinology Center, University of Washington Medical Center, Seattle, Washington. PATIENT(S) Four hundred forty-one patients undergoing their first attempt at IVF-ICSI from January 1, 1999, to May 21, 2001. INTERVENTION(S) Each ICSI procedure for an individual patient was performed by a single operator. Sperm parameters, oocyte age, culture condition, ICSI technique, and ICSI operator were assessed as variables influencing the fertilization rate. We also assessed the impact of patient age, serum E(2) concentration on the day of hCG administration, embryo quality, and number of embryos transferred on the probability of achieving a clinical pregnancy. MAIN OUTCOME MEASURE(S) Fertilization rate and clinical pregnancy. RESULT(S) The 2 pronuclei (2PN) rate was significantly correlated with sperm motility, and there were significant differences in the 2PN rates among the ICSI operators. There was no difference in the 2PN rate among different sperm types or among the eight laboratory incubators or whether the eggs were cultured individually or in groups. Patient age, serum E(2) concentration on the day of hCG administration, embryo quality, and number of embryos transferred were all statistically significant predictors of clinical pregnancy. CONCLUSION(S) In our program, sperm motility and ICSI operator are the two most important predictors for the ICSI fertilization rate in vitro. Patient age, serum E(2) concentration on the day of hCG administration, embryo quality, and number of embryos transferred were all statistically significant predictors of clinical pregnancy.

Practice patterns and outcomes with the use of single embryo transfer in the United States

OBJECTIVE To evaluate factors associated with the use of elective single embryo transfer (eSET) and its effect on assisted reproductive technology (ART) outcome. DESIGN Historical cohort. SETTING Clinic-based data. PATIENT(S) A total of 69,028 ART cycles of autologous fresh embryo transfers with additional embryos cryopreserved during the same cycle performed during 2004-06 and reported to the Society for Assisted Reproductive Technology Clinic Outcome Reporting System database. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Factors associated with the number of embryos transferred, and the odds of pregnancy, live birth, and multiple-infant live birth by number of embryos transferred as adjusted odds ratios (AORs). RESULT(S) Single embryo transfer was used more with uterine factor (AOR 1.76) and less with male factor, endometriosis, or tubal factor (AOR 0.81, 0.72, 0.83, respectively). Compared with women aged <30 years, eSET was used less among women aged 35-39 years and > or =40 years (AOR 0.74 and 0.39, respectively). Compared with White women, eSET was used more with Asian (AOR 1.52) and less with Black or Hispanic women (AOR 0.73 and 0.67, respectively). Compared with eSET, the likelihood of pregnancy, live birth, or multiple-infant live birth was more likely with two embryos (AOR 1.33, 1.34, and 27.4, respectively). CONCLUSION(S) Elective SET, used more for younger women with specific diagnoses, is associated with slightly reduced likelihood of a live birth but much reduced likelihood of multiples.