Emanuela Lazzaroni

A pilot cohort study of granulocyte colony-stimulating factor in the treatment of unresponsive thin endometrium resistant to standard therapies

STUDY QUESTION Is thin endometrium unresponsive to standard treatments expandable by intrauterine perfusion with granulocyte colony-stimulating factor (G-CSF)? SUMMARY ANSWER This cohort study is supportive of the effectiveness of G-CSF in expanding chronically unresponsive endometria. WHAT IS KNOWN ALREADY In a previous small case series, we reported the successful off-label use of G-CSF in four consecutive patients, who had previously failed to expand their endometria beyond 6.9 mm with the use of standard treatments. STUDY DESIGN, SIZE AND DURATION In a prospective observational cohort pilot study over 18 months, we described 21 consecutive infertile women with endometria <7 mm on the day of hCG administration in their first IVF cycles at our center. All previous cycles using traditional treatments with estradiol, sildenafil citrate (Viagra™) and/or beta-blockers had been unsuccessful. G-CSF (Nupogen™) was administered per intrauterine catheter by slow infusion before noon on the day of hCG administration. If the endometrium had not reached at least a 7-mm within 48h, a second infusion was given following oocyte retrieval. Primary and secondary main outcomes were an increase in endometrial thickness and clinical pregnancy, respectively. Endometrial thickness was assessed by vaginal ultrasound at the most expanded area of the endometrial stripe. PARTICIPANTS/MATERIALS, SETTINGS AND METHOD This study was uncontrolled, each patient serving as her own control in a prospective evaluation of endometrial thickness. The mean ± SD age of the cohort was 40.5 ± 6.6 years, gravidity was 1.8 ± 2.1 (range 0-7) and parity was 0.4 ± 1.1 (range 0-4); 76.2% of women had, based on age-specific FSH and anti-Müllerian hormone, an objective diagnosis of diminished ovarian reserve and had failed 2.0 ± 2.1 prior IVF cycles elsewhere. MAIN RESULTS AND THE ROLE OF CHANCE With 5.2 ± 1.9 days between G-CSF perfusions and embryo transfers, endometrial thickness increased from 6.4 ± 1.4 to 9.3 ± 2.1 mm (P < 0.001). The Δ in change was 2.9 ± 2.0 mm, and did not vary between conception and non-conception cycles. A 19.1% ongoing clinical pregnancy rate was observed, excluding one ectopic pregnancy. LIMITATIONS AND REASONS FOR CAUTION Small sample size (but a highly selected patient population) in an uncontrolled cohort study and in unselected first IVF cycles at our center. WIDER IMPLICATIONS OF THE FINDINGS This pilot study supports the utility of G-CSF in the treatment of chronically thin endometrium and suggests that such treatment will, in very adversely affected patients, result in low but very reasonable clinical pregnancy rates. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Foundation for Reproductive Medicine, New York, New York, USA, a not-for-profit research foundation and intramural grants from the Center for Human Reproduction (CHR)-New York. N.G. and D.H.B. are members of the board of the Foundation for Reproductive Medicine. N.G. is owner of CHR-New York, where the study was conducted. N.G. and D.H.B. have been recipients of research awards, travel grants and speaker honoraria from various pharmaceutical and medical device companies. None of these companies was, however, in any way associated with the materials and the manuscript presented here. N.G. and D.H.B. are listed as co-inventors on a number of awarded and still pending U.S. patents, none related to the materials presented here. N.G. is on the board of a medically related company, not in any way associated with the data presented here.

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.

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.

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 17β-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.

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 genes importance for reproductive success.

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

BackgroundLow 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.Methods322 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.ResultsWomen 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.ConclusionsIn 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.