Michael Gal

DHEA supplementation may improve IVF outcome in poor responders: a proposed mechanism.

OBJECTIVE Dehydroepiandrosterone (DHEA) supplementation for poor responders may improve ovarian response and IVF treatment outcome. This study aimed to determine the mechanism of action of DHEA, and specifically, the stage of folliculogenesis influenced by DHEA. STUDY DESIGN This is a prospective, self-controlled study of poor responders to IVF treatment, comparing day 3 biochemical (anti-Mullerian hormone (AMH), inhibin B and FSH) and ultrasound (antral follicle count (AFC)) ovarian reserve markers and IVF treatment outcome before and after DHEA supplementation of at least 3 months duration. RESULTS Thirty-two women were included. Following DHEA, there was a significant increase in AFC (P=0.0003) without significant changes in the baseline biochemical parameters AMH, inhibin B, or FSH. The enhanced response comprised increased peak estradiol levels (P=0.0005), number of follicles >15 mm, oocytes, MII oocytes and embryos (P=0.004, P=0.00001, P=0.0004 and P=0.0006, respectively) and oocytes number/total FSH dose (P=0.0009). The proportion of cancelled cycles due to very poor response decreased significantly (P=0.02). CONCLUSIONS DHEA does not appear to exert influence via recruitment of pre-antral or very small antral follicles (no change in AMH and inhibin B) but rather by rescue from atresia of small antral follicles (increased AFC).

Low dose ketoconazole attenuates serum androgen levels in patients with polycystic ovary syndrome and inhibits ovarian steroidogenesis in vitro.

OBJECTIVE To investigate the effects of a low-dose ketoconazole on ovarian steroidogenesis and on serum androgen levels in polycystic ovary syndrome (PCOS). DESIGN In vitro, human granulosa-luteal cells were incubated with ketoconazole and radiolabeled steroid substrates, to follow their metabolic fate by thin-layer chromatography analysis. In vivo, normally cycling women (n = 7) in their luteal phase were administered one tablet of 200 mg ketoconazole at 8 A.M. Serum steroid levels, sampled basally and at 12 P.M., 4 P.M., and 8 A.M. the next morning, were compared with untreated control group (n = 7) values. Polycystic ovary syndrome women (n = 11) were similarly administered ketoconazole 6 to 10 days after occurrence of spontaneous menses. Adrenal origin of hyperandrogenemia was excluded by stimulation with ACTH and a normal basal DHEAS. The steroid diurnal variation was determined in the same patients a day before treatment. RESULTS In vitro, ketoconazole selectively inhibited the key steroidogenic cytochromes, namely P450scc, P45017 alpha, and P450arom (IC50 = 0.5 to 1.0 microgram/mL). In vivo, in the luteal phase, ketoconazole transiently decreased serum values (mean +/- SE) of E2 (19.2% +/- 2.1%) and P (38.3% +/- 8.5%) within 4 to 8 hours. The same low-dose ketoconazole, administered to PCOS women, decreased serum values of androstenedione (17.6% +/- 4.7%), T (24.6% +/- 7.6%), and free T (30.7% +/- 7.7%). In contrast, 17 alpha-hydroxyprogesterone increased concomitantly (78.5% +/- 10.8%), suggesting a greater suppressibility of the P45017 alpha lyase activity. The E2 levels in PCOS patients were slightly elevated (29.1% +/- 5.6%), resulting in a 1.7- to 2.3-fold increase of the E2:T ratio. CONCLUSIONS These findings suggest that a low-dose ketoconazole may facilitate a decreased intraovarian T:E2 ratio, which may prove favorable for follicular maturation in PCOS.

Effect of ketoconazole on steroidogenic human granulosa-luteal cells in culture.

Ketoconazole (KCZ), a widely used antifungal drug, has been reported in humans to inhibit adrenal and testicular steroidogenesis by interfering with the cytochrome P-450-dependent enzymes. The purpose of this study was to investigate the drug effect on steroidogenic human granulosa-luteal cells, obtained by follicular aspiration from mature follicles of gonadotropin-treated women. Cells were cultured in long-term monolayers, and the steroid production was assayed by radioimmunoassay. A profound inhibition of ovarian cell secretion of progesterone (P), testosterone (T) and estradiol was found. At a low concentration (5 micrograms/ml), KCZ failed to inhibit the conversion of pregnenolone to P, mediated by the non-cytochrome 3 beta-hydroxysteroid dehydrogenase-isomerase enzyme (3 beta-HSDH). At a similar concentration, P secretion by human chorionic gonadotropin (hCG; 100 mIU/ml) -treated cells was decreased by 68% (P less than 0.001) and therefore, an inhibitory effect of KCZ on the cholesterol side-chain cleavage enzyme (P-450SCC) was assumed. A similar marked inhibitory effect (81%) (P less than 0.001) on T secretion was observed for hCG-stimulated cells given pregnenolone as substrate. The P-450 aromatase was profoundly inhibited (86%) (P less than 0.001) in a reversible manner, by a similar concentration (5 micrograms/ml) of KCZ. These findings suggest that KCZ has the capability to suppress human ovarian steroidogenesis similarly as in testis and adrenal.

Ovarian stimulation for oocyte cryopreservation for prevention of age-related fertility loss: one in five is a low responder

Abstract Oocyte cryopreservation for age-related fertility loss is gaining interest considering the tendency to postpone motherhood in many societies. Little is currently known about the actual efficiency of this approach. We aimed to explore ovarian response of presumably fertile women undergoing in vitro fertilization for this indication. A total of 105 women underwent 151 stimulation cycles at mean age 37.7 ± 2.4. None had known infertility. Mean daily starting FSH dose was 371 ± 110 (225–600). Mean number of mature oocytes cryopreserved at the first completed cycle was 9.7 ± 7.5 (0–43). However, 21% of started cycles were either cancelled before egg retrieval or resulted in 0–3 mature oocytes retrieved. Therefore, women considering oocyte cryopreservation for prevention of age-related fertility decline should be encouraged to perform this procedure at younger age than, preferably before 35.

Attenuation of ovarian response by low-dose ketoconazole during superovulation in patients with polycystic ovary syndrome.

OBJECTIVE To investigate the clinical efficacy of mild inhibition of ovarian steroidogenesis by very low-dose ketoconazole during induction of ovulation in patients with polycystic ovary syndrome (PCOS). DESIGN Prospective, randomized, cross-controlled study in consecutive cycles. SETTING Large tertiary care center. PATIENT(S) Eighteen patients with PCOS undergoing hMG superovulation with or without ketoconazole. INTERVENTION(S) A fixed hMG dosage was initiated on cycle days 5-9 in both of the study cycles. Further hMG adjustment was done according to serum E2 levels and follicular measurements. Ketoconazole was administered in one of the cycles by two protocols. MAIN OUTCOME MEASURE(S) Serum E2 and P levels, lead follicles, pregnancy rate, and development of ovarian hyperstimulation syndrome. RESULT(S) Although higher daily hMG doses were needed in cycles with ketoconazole compared with cycles without the drug, the peak E2 levels were substantially lower in the ketoconazole cycles. Although the number of lead follicles did not differ between treatments, the addition of ketoconazole significantly reduced the number of hyperstimulated cycles. Consequently, the cancellation rate dropped dramatically, thus yielding a higher pregnancy rate per patient in the ketoconazole protocols. CONCLUSION(S) Use of a very low dose of ketoconazole during ovulation induction effectively attenuates ovarian steroidogenesis in patients with PCOS. This effect may serve as an adjunct to better control the ovarian response in women who are prone to hyperstimulated cycles.

Selective Inhibition of Steroidogenic Enzymes by Ketoconazole in Rat Ovary Cells

Objective Ketoconazole (KCZ) is an anti-fungal agent extensively used for clinical applications related to its inhibitory effects on adrenal and testicular steroidogenesis. Much less information is available on the effects of KCZ on synthesis of steroid hormones in the ovary. The present study aimed to characterize the in situ effects of KCZ on steroidogenic enzymes in primary rat ovary cells. Methods Following the induction of folliculogenesis in gonadotropin treated rats, freshly prepared ovarian cells were incubated in suspension for up to four hours while radiolabeled steroid substrates were added and time dependent generation of their metabolic products was analyzed by thin layer chromatography (TLC). Results KCZ inhibits the P450 steroidogenic enzymes in a selective and dose dependent manner, including cholesterol side-chain cleavage cytochrome P450 (CYP11A1/P450scc), the 17α-hydroxylase activity of CYP17A1/P450c17, and CYP19A1/P450arom, with IC50 values of 0.3, 1.8, and 0.3 μg/mL (0.56, 3.36, and 0.56 μM), respectively. Unaffected by KCZ, at 10 μg/mL, were the 17,20 lyase activity of CYP17A1, as well as five non-cytochrome steroidogenic enzymes including 3β-hydroxysteroid dehydrogenase-δ5-4 isomerase type 1 (3βHSD1), 5α-reductase, 20α-hydroxysteroid dehydrogenase (20α-HSD), 3α-hydroxysteroid dehydrogenase (3α-HSD), and 17β-hydroxysteroid dehydrogenase type 1 (17HSD1). Conclusion These findings map the effects of KCZ on the ovarian pathways of progestin, androgen, and estrogen synthesis. Hence, the drug may have a potential use as an acute and reversible modulator of ovarian steroidogenesis in pathological circumstances.

Preconception screening for cytomegalovirus: an effective preventive approach.

Congenital cytomegalovirus (CMV) is the leading infectious cause of sensorineural hearing loss and delayed psychomotor development. Viral transmission to the fetus is far more likely to occur following a primary than a secondary maternal infection. Primary prevention seems to be the best means to reduce the burden of congenital CMV due to the lack of treatment options during pregnancy. We evaluated this approach on a cohort of 500 women planning pregnancy who attended our fertility clinic. Of the 444 who underwent CMV screening, 18 (4.1%) had positive IgM serology for CMV; of these, IgG avidity was high in 12 (remote infection) and low in 6 (recent infection). The latter were advised to delay pregnancy. All women who were seroimmune for CMV (366/444, 82.4%), including the 12 with remote infection, continued fertility treatment. The remaining patients (72/444, 16.2%), who were not immune to CMV at the initial screen, were advised to minimize CMV exposure by improving personal hygiene and to continue fertility treatment. None of the 69/72 (95.8%) women who were followed for one year were infected with CMV. Cytomegalovirus testing and counselling at preconception seemed effective in reducing CMV exposure in pregnancy.

HP1BP3 expression determines maternal behavior and offspring survival.

Maternal care is an indispensable behavioral component necessary for survival and reproductive success in mammals, and postpartum maternal behavior is mediated by an incompletely understood complex interplay of signals including effects of epigenetic regulation. We approached this issue using our recently established mice with targeted deletion of heterochromatin protein 1 binding protein 3 (HP1BP3), which we found to be a novel epigenetic repressor with critical roles in postnatal growth. Here, we report a dramatic reduction in the survival of pups born to Hp1bp3−/− deficient mouse dams, which could be rescued by co‐fostering with wild‐type dams. Hp1bp3−/− females failed to retrieve both their own pups and foster pups in a pup retrieval test, and showed reduced anxiety‐like behavior in the open‐field and elevated‐plus‐maze tests. In contrast, Hp1bp3−/− females showed no deficits in behaviors often associated with impaired maternal care, including social behavior, depression, motor coordination and olfactory capability; and maintained unchanged anxiety‐associated hallmarks such as cholinergic status and brain miRNA profiles. Collectively, our results suggest a novel role for HP1BP3 in regulating maternal and anxiety‐related behavior in mice and call for exploring ways to manipulate this epigenetic process.

Ketoconazole inhibits ovulation as a result of arrest of follicular steroidogenesis in the rat ovary.

Objective Ketoconazole (KCZ) is a known inhibitor of steroidogenic P450 enzymes in the adrenal cortex and the gonads. Previous studies examined the potential clinical use of KCZ for attenuation of ovarian response to gonadotropin treatments. This study aimed to use the superovuating rat model to explore the effect of KCZ on ovarian steroidogenesis, follicular function, and development toward ovulation. Methods Prepubertal rats were treated with equine chorionic gonadotropin (eCG)/human CG (hCG) resulting in multiple follicular development and ovulation. The effect of KCZ on this model was examined by administration of KCZ-gel formula and subsequent analyses of ovarian steroidogenesis, rate of ovulation, morphometric assessments of follicular parameters, and cell-specific steroidogenic maturation of the treated ovaries. Results When applied shortly before gonadotropin stimulation, KCZ markedly reduced ovarian progesterone, androstenedione, and estradiol levels down to 18.7, 36.5, and 19.0%, respectively (P < 0.001). A single KCZ-gel administration of 6, 12, and 24 mg/rat resulted in reduction of ovulated ova/ovary down to 8.6 ± 4.9, 5.1 ± 4.3, and 2.4 ± 3.2, respectively, as compared to 13.6 ± 4.4 ova found in the oviduct of control-gel-injected animals (P < 0.001). An alternative protocol made use of small KCZ doses injected in non-gel formula (5 mg/dose/8 hours), commenced with the eCG administration and terminated 24 hours later; this treatment readily inhibited the ovulation rates to 6.6 ± 6.6 as compared to 16.5 ± 4.1 ova/ovary in the control group (P < 0.01). By contrast, KCZ failed to inhibit ovulation if administered 24 hours after eCG injection. Anovulation by KCZ resulted from arrest of follicular development at the stage of 800-840 μm Graafian follicles as compared to 920 μm of peri-ovulatory follicles (OFs) observed in the control group, P = 0.029. In addition, absence of CYP11A1 expression was evident in the granulosa cell layers of the growth-arrested follicles, which also lacked mucified mature cumulus cell complexes. Conclusion These results suggest that KCZ-mediated inhibition of follicular maturation probably results from impaired steroidogenesis at early phase of follicular development toward ovulation. Hence, attenuation of folliculogenesis by KCZ may be harnessed to modulate gonadotropin-ovarian stimulation in fertility treatments.

Comparison of the effect of sperm collection by condom or masturbation on the outcome of in-vitro fertilization.

Improved sperm indices have been reported when semen produced on oocyte pick-up (OPU) day is collected with a condom compared with by masturbation among both patients with normozoospermia and those with sperm deficiencies [1, 2]. However, potential benefits of sperm collection by condom have not been evaluated in an in vitro fertilization (IVF) setting. The present study retrospectively investigated the effect of the sperm collection by condom or masturbation on the outcome of OPU-embryo transfer (OPU-ET) cycles. Pregnancy and fertilization rates were compared based on the type of oocyte insemination (IVF-insemination or intracytoplasmic sperm injection), and across sperm deficiencies. This article is protected by copyright. All rights reserved.