Aging-related premature luteinization of granulosa cells is avoided by early oocyte retrieval.

Why IVF pregnancy rates decline sharply after age 43 is unknown. In this study, we compared granulosa cell (GC) function in young oocyte donors (n=31, ages 21-29), middle-aged (n=64, ages 30-37) and older infertile patients (n=41, ages 43-47). Gene expressions related to gonadotropin activity, steroidogenesis, apoptosis and luteinization were examined by real-time PCR and western blot in GCs collected from follicular fluid. FSH receptor (FSHR), aromatase (CYP19A1) and 17ß-hydroxysteroid dehydrogenase (HSD17B) expression were found down regulated with advancing age, while LH receptor (LHCGR), P450scc (CYP11A1) and progesterone receptor (PGR) were up regulated. Upon in vitro culture, GCs were found to exhibit lower proliferation and increased apoptosis with aging. While FSH supplementation stimulated GCs growth and prevented luteinization in vitro. These observations demonstrate age-related functional declines in GCs, consistent with premature luteinization. To avoid premature luteinization in women above age 43, we advanced oocyte retrieval by administering human chorionic gonadotropin at maximal leading follicle size of 16  mm (routine 19-21  mm). Compared to normal cycles in women of similar age, earlier retrieved patients demonstrated only a marginal increase in oocyte prematurity, yet exhibited improved embryo numbers as well as quality and respectable clinical pregnancy rates. Premature follicular luteinization appears to contribute to rapidly declining IVF pregnancy chances after age 43, and can be avoided by earlier oocyte retrieval.

Effect of inter-cycle interval on oocyte production in humans in the presence of the weak androgen DHEA and follicle stimulating hormone: a case-control study.

BACKGROUND: In various animal models androgens have been demonstrated to enhance follicle stimulating hormone (FSH) activity on granulosa cells during small growing follicle stages. To assess whether similar synergism may also exist in humans we investigated women on androgen (dehydroepiandrosterone, DHEA) supplementation with varying concomitant FSH exposure. METHODS: In a case controlled cohort study we determine if time interval between IVF cycles of IVF treatment with FSH had an effect on ovarian response to ovulation induction in women supplemented with DHEA. Among 85 women with known low functional ovarian reserve (LFOR), supplemented with DHEA, and undergoing at least 3 consecutive IVF cycles, 68 demonstrated short (<120 days) intervals between repeated cycles (Group 1) and were, therefore, considered to have consistent FSH exposure. In contrast 17 women (Group 2) demonstrated long (>=120 days) intervals between repeated cycles and, therefore, were considered to demonstrate inconsistent FSH exposure. Trends in oocyte yields were compared between these groups, utilizing mixed model repeated measures ANOVA, adjusted for initial age and FSH dose. RESULTS: Only women in Group I demonstrated a linear increase in oocyte yields across their three cycles of treatments (F=7.92; df 1, 68.6; p=0.017). Moreover, the analysis revealed a significant interaction between the two patient groups and cycle number for retrieved oocytes (F=6.32, df=2, 85.9, p=0.003). CONCLUSIONS: This study offers preliminary confirmatory evidence that repeated short interval exposure to androgens in combination with FSH improves human FOR. A higher level of evidence will require prospectively randomized studies.

A randomized clinical trial of endometrial perfusion with granulocyte colony-stimulating factor in in vitro fertilization cycles: impact on endometrial thickness and clinical pregnancy rates.

OBJECTIVE: To investigate whether granulocyte colony-stimulating factor (G-CSG) affects endometrial thickness, implantation rates, and clinical pregnancy rates in routine, unselected IVF cycles. DESIGN: Registered, individually randomized, two-group, parallel double-blinded placebo-controlled clinical trial. SETTING: Academically affiliated private clinical and research center. PATIENT(S): 141 consecutive, unselected, consenting women with no history of renal disease, sickle cell disease, or malignancy who were undergoing IVF. INTERVENTION(S): Sealed, numbered, opaque envelopes assigned 73 patients to receive G-CSF (Filgrastim, Amgen, 300 µg/1.0 mL) and 68 to receive placebo (saline). MAIN OUTCOME MEASURE(S): Endometrial thickness, clinical pregnancy, and embryo implantation rates. RESULT(S): The mean age for the whole study group was 39.59 ± 5.56 years (G-CSF: 39.79 ± 5.13 years; placebo: 39.38 ± 6.03 years). Endometrial thickness statistically significantly increased over the 5-day observation period for the whole group by approximately 1.36 mm. The increase in the G-CSF group was not statistically significantly different from the control group. Statistical models looking at treatment effects on clinical pregnancy and implantation rates demonstrated no effect of G-CSF treatment. There were no adverse events for either treatment group. CONCLUSION(S): In normal IVF patients, G-CSF does not affect endometrial thickness, implantation rates, or clinical pregnancy rates. Because these results were obtained in an older patient population, they may not necessarily apply to younger women. CLINICAL TRIAL REGISTRATION NUMBER: NCT01202656.

FMR1-dependent variability of ovarian aging patterns is already apparent in young oocyte donors.

BACKGROUND: Hypothesizing that redundant functional ovarian reserve (FOR) at young ages may clinically obfuscate prematurely diminished FOR (PDFOR), we investigated in young oocyte donors genotypes and sub-genotypes of the FMR1 gene, in prior studies associated with specific ovarian aging patterns, and determined whether they already at such young age were associated with variations in ovarian reserve (OR). We also investigated racial as well as FMR1 associations with menarcheal age in these donors. METHODS: In a cohort study we investigated 157 oocyte donor candidates and, based on the 95% CI of AMH, divided them into normal age-specific (AMH greater or equal to 2.1 ng/mL; n = 121) and PDFOR (AMH < 2.1 ng/mL; n = 36). We then assessed associations between numbers of trinucleotide repeat (CGGn) on the FMR1 gene and FOR (based on anti-Müllerian hormone, AMH). RESULTS: FMR1 did not associate with AMH overall. Amongst 36 donors with PDFOR, 17 (42%) presented with at least one low (CGGn < 26 ) allele. Remaining donors with normal FOR presented with significantly more CGGn greater or equal to 26 (73.6% vs. 26.4%; P = 0.024) and higher AMH (P = 0.012). This finding was mostly the consequence of interaction between FMR1 (CGGn < 26 vs. CGGn greater or equal to 26) and race (P = 0.013), with Asians most responsible (P = 0.009). Menarcheal age was in donors with normal FOR neither associated with race nor with FMR1 status. In donors with PDFOR race was statistically associated with CGGn (P = 0.018), an association primarily based on significantly delayed age of menarche in African donors with CGGn < 26 in comparison to African donors with CGGn greater or equal to 26 (P = 0.019), and Caucasian (P = 0.017) and Asian donors (P = 0.025) with CGGn < 26. CONCLUSIONS: CGGn on FMR1 already at young ages affects FOR, but is clinically apparent only in cases of PDFOR. Screening for low FMR1 CGGn < 26 at young age, thus, appears predictive of later PDFOR.

Is androgen production in association with immune system activation potential evidence for existence of a functional adrenal/ovarian autoimmune system in women?

BACKGROUND: Low functional ovarian reserve (FOR) is at all ages associated with low testosterone (T) levels. Causes are, however, unknown. We, therefore, investigate whether androgens with low FOR are associated with non-specific immune system activation. METHODS: 322 infertile women with low and normal FOR (controls) were assessed with a broadly based immune profile, which in previous studies has proven effective in differentiating infertile patients with and without immune system activation. Patients were either immune-positive (greater than or equal to one positive tested parameter) or immune negative (no positive test). 135 suffered from prematurely diminished FOR (POA/OPOI; < age 38), 155 from physiologic diminished FOR due to age (DOR; > age 40), and 32 were controls (< age 38 with normal age-specific FOR). Prevalence of immune-positive vs. negative was assessed in all 3 patient groups. RESULTS: Women with immune abnormalities, overall, demonstrated higher total T (TT, P = 0.004) and free T (FT, P < 0.001) levels than those without. The three clinical and two immunologic-defined patient groups demonstrated significant statistical interaction in mean TT (P = 0.008), with mean TT and FT in women with positive immune findings being significantly higher in control than in POA/OPOI and physiologic DOR patients (all 4 differences P < 0.001). No such differences between the three groups were seen in women without immune abnormalities. CONCLUSIONS: In this study we used a definition of immune-positivity, which favors sensitivity over specificity, resulting in significant numbers of false-positives but likely only few false-negatives. The study allows suggesting the possibility of an immune system-derived androgen-production factor (APF), which maintains normal androgen levels but is deficient in women with low FOR and immune system inactivity. Existence of such an APF would suggest the presence of a still unknown functional adrenal autoimmune system.

Clinical relevance of combined FSH and AMH observations in infertile women.

CONTEXT: FSH and anti-Müllerian hormone (AMH) are, individually, widely used to assess functional ovarian reserve (FOR) but demonstrate discrepancies in efficacy. How predictive they are combined is unknown. OBJECTIVE: The purpose of this study was to assess predictive values of different FSH and AMH combinations on in vitro fertilization (IVF). DESIGN AND SETTING: FSH and AMH levels in patients were categorized as low, normal, and high, based on age-specific 95% confidence intervals. This allowed for establishment of nine combinations of low, normal, or high FSH/AMH patient categories. With use of various statistical methods, patients in individual categories were then compared in outcomes. PATIENTS: We investigated 544 consecutive infertility patients in their first IVF cycles. INTERVENTIONS: IVF cycles were managed. MAIN OUTCOME MEASURES: Oocyte yields and implantation and pregnancy rates, adjusted for age and fragile X mental retardation 1 (FMR1) genotypes/subgenotypes, were measured. RESULTS: The most notable repeated finding was a strong statistical association of the FSH/AMH high/high category (characterized by abnormally high FSH and AMH levels) with favorable IVF outcomes compared with outcomes for other FSH/AMH variations (4.34 times odds of high oocyte yields and 1.93 times odds of clinical pregnancy). Addition of age to the model only minimally further improved the odds of pregnancy to 2.03 times. The positive association with high oocyte yields, however, turned negative (0.75 times lower yields) with addition of FMR1 to the model for women with FSH/AMH high/high and the het-norm/low FMR1 subgenotype compared with women with the norm FMR1 genotype and other FSH/AMH categories. CONCLUSIONS: In the absence of het-norm/low FMR1, abnormally high FSH and AMH, a seemingly contradictory combination, reflects highly beneficial outcomes in IVF compared with the other FSH/AMH categories, suggesting greater importance of FSH in early follicle maturation than currently recognized. The study also confirms adverse outcome effects of het-norm/low FMR1 and, therefore, the gene's importance for reproductive success.

Hypoandrogenism in association with diminished functional ovarian reserve.

STUDY QUESTION: Is diminished functional ovarian reserve (DFOR) associated with low androgen levels? SUMMARY ANSWER: Low androgen levels are associated with DFOR at all ages. WHAT IS KNOWN ALREADY: Androgen supplementation via dehydroepiandrosterone (DHEA) has been reported to improve functional ovarian reserve (FOR); pregnancy rates in IVF cycles are associated with how well DHEA converts to testosterone (T); and androgen effects through the androgen receptor have been demonstrated in mice to beneficially affect early stages of follicle maturation. STUDY DESIGN, SIZE, DURATION: In a controlled cohort study we investigated consecutive women presenting to our center with two forms of DFOR, premature ovarian aging/occult primary ovarian insufficiency (POA/OPOI) and physiologic diminished ovarian reserve (DOR). As controls for POA/OPOI patients, infertile women with normal age-specific FOR were recruited. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: The study involved 140 women with POA/OPOI, defined as age <38 years and abnormally low FOR by age-specific FSH and/or anti-Müllerian hormone (AMH), 166 women with DOR, defined as women age >40 years. Forty-nine control patients <38 years demonstrated normal FOR by FSH and/or AMH. In all three patient groups dehydroepiandrosterone (DHEA), DHEA-sulfate (DHEAS), total testosterone (TT) and free testosterone (FT) at the time of initial presentation to our fertility center were assessed. In a small subgroup of women early morning cortisol levels were also assessed. MAIN RESULTS AND THE ROLE OF CHANCE: DHEAS marginally varied between the three groups (P = 0.04), with older women with DOR actually demonstrating higher levels than controls (P = 0.03). TT differed between the three groups more profoundly (P = 0.005), with women with POA/OPOI demonstrating significantly lower levels than controls (P = 0.009). Adjustment for body mass index, age and race in principle maintained observed differences in TT between groups, while adjustment for FMR1 (fragile X mental retardation 1) genotypes/sub-genotypes eliminated all differences. All three patient groups demonstrated low morning cortisol levels. LIMITATIONS, REASONS FOR CAUTION: While results support lower androgen levels in women with DOR, and even more so in women with POA/OPOI, presented data should be viewed as preliminary, considering the known variability of androgen levels and the small number of women in whom morning cortisol levels were available. WIDER IMPLICATIONS OF THE FINDINGS: Especially at young ages DFOR appears associated with significant hypoandrogenism (low T) in comparison with young control patients with normal FOR, raising the question whether this hypoandrogenism originates in adrenals or ovaries. POA/OPOI, thus, phenotypically mimics the polycystic ovary syndrome, where similar questions arise, though in regard to hyperandrogenism. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the Foundation for Reproductive Medicine, a not-for-profit medical research foundation and intramural funds from the Center for Human Reproduction. N.G. and D.H.B are members of the Board of the Foundation for Reproductive Medicine. N.G., A.W. and D.H.B. received research support, lecture fees and travel support from a variety of pharmaceutical and medical device companies, none in any way related to the issues discussed in this manuscript. N.G. and D.H.B. are listed as co-inventors on two, already granted US user patents, which claim therapeutic benefits from DHEA supplementation in women with DFOR and DOR: both authors are also listed on additional pending patents in regard to DHEA supplementation and on pending patents, claiming diagnostic and therapeutic benefits from the determination of CGG repeats on the FMR1 gene. N.G. is the owner of the Center for Human Reproduction, where this research was performed.

Starting and resulting testosterone levels after androgen supplementation determine at all ages in vitro fertilization (IVF) pregnancy rates in women with diminished ovarian reserve (DOR).

PURPOSE: To investigate whether androgen conversion rates after supplementation with dehydroepiandrosterone (DHEA) differ, and whether differences between patients with diminished ovarian reserve (DOR) are predictive of pregnancy chances in association with in vitro fertilization (IVF). METHODS: In a prospective cohort study we investigated 213 women with DOR, stratified for age (≤ 38 or >38 years) and ovarian FMR1 genotypes/sub-genotypes. All women were for at least 6 weeks supplemented with 75 mg of DHEA daily prior to IVF, between initial presentation and start of 1st IVF cycles. Levels of DHEA, DHEA-sulfate (DHEAS), total T (TT) and free T (FT) at baseline ((BL)) and IVF cycle start ((CS)) were then compared between conception and non-conception cycles. RESULTS: Mean age for the study population was 41.5 ± 4.4 years. Forty-seven IVF cycles (22.1 %) resulted in clinical pregnancy. Benefits of DHEA on pregnancy rates were statistically associated with efficiency of androgen conversion from DHEA to T and amplitude of T gain. Younger women converted significantly more efficiently than older females, and selected FMR1 genotypes/sub-genotypes converted better than others. FSH/androgen and AMH/androgen ratios represent promising new predictors of IVF pregnancy chances in women with DOR. CONCLUSIONS: DOR at all ages appears to represent an androgen-deficient state, benefitting from androgen supplementation. Efficacy of androgen supplementation with DHEA, however, varies depending on female age and FMR1 genotype/sub-genotype. Further clarification of FMR1 effects should lead to better individualization of androgen supplementation, whether via DHEA or other androgenic compounds.