Erkan Buyuk

Embryo development after heterotopic transplantation of cryopreserved ovarian tissue

BACKGROUND Cancer treatments, including chemotherapy, radiotherapy, and radical surgery, can induce premature menopause and infertility in hundreds of thousands of women of reproductive age every year. One of the ways to possibly preserve fertility before these treatments is to cryopreserve ovarian tissue for later transplantation. We aimed to restore fertility by cryopreservation and transplantation of ovarian tissue. METHODS Ovarian tissue was cryopreserved from a 30-year-old woman with breast cancer before chemotherapy-induced menopause, and this tissue was transplanted beneath the skin of her abdomen 6 years later. FINDINGS Ovarian function returned in the patient 3 months after transplantation, as shown by follicle development and oestrogen production. The patient underwent eight oocyte retrievals percutaneously and 20 oocytes were retrieved. Of the eight oocytes suitable for in-vitro fertilisation, one fertilised normally and developed into a four-cell embryo. INTERPRETATION Fertility and ovarian endocrine function can be preserved in women by long-term ovarian tissue banking.

Fertility Preservation in Breast Cancer Patients: A Prospective Controlled Comparison of Ovarian Stimulation With Tamoxifen and Letrozole for Embryo Cryopreservation

Purpose To develop safe ovarian stimulation methods to perform in vitro fertilization (IVF) in breast cancer patients who wish to preserve their fertility via embryo cryopreservation before chemotherapy. Patients and Methods Sixty women (age range, 24 to 43 years) with breast cancer were prospectively studied. Twenty-nine patients underwent 33 ovarian stimulation cycles with either tamoxifen 60 mg/d alone (Tam-IVF) or in combination with low-dose follicle-stimulating hormone (TamFSH-IVF) or letrozole 5 mg in combination with FSH (Letrozole-IVF). After IVF, all resultant embryos were cryopreserved to preserve fertility. Recurrence rates were compared with controls (n 31) who elected not to undergo IVF. Results Compared with Tam-IVF, both TamFSH-IVF and Letrozole-IVF patients had greater numbers of follicles (2 0.3 v 6 1 and 7.8 0.9, respectively; P .0001), mature oocytes (1.5 0.3 v 5.1 1.1 and 8.5 1.6, respectively; P .001), and embryos (1.3 0.2 v 3.8 0.8 and 5.3 0.8, respectively; P .001). Peak estradiol (E2) levels were lower with Letrozole-IVF and Tam-IVF compared with TamFSH-IVF. After 554 31 days (range, 153 to 1,441 days) of follow-up, cancer recurrence rate was similar between IVF and control patients (three of 29 v three of 31 patients, respectively; hazard ratio, 1.5; 95% CI, 0.29 to 7.4), and this estimate was not affected by cancer stage. Conclusion The combination of low-dose FSH with tamoxifen (TamFSH-IVF) or letrozole (Letrozole-IVF) results in higher embryo yield compared with Tam-IVF. Recurrence rates do not seem to be increased, but the letrozole protocol may be preferred because it results in lower peak E2 levels.

A technique for transplantation of ovarian cortical strips to the forearm

OBJECTIVE To describe a forearm heterotopic ovarian transplantation technique. DESIGN Case study. SETTING Academic medical center. PATIENT(S) One patient with stage IIIB squamous cell cervical carcinoma and one patient with recurrent benign ovarian cysts. INTERVENTION(S) Preparation of thin ovarian cortical slices and transplantation under the skin of the forearm. MAIN OUTCOME MEASURE(S) Follicular development and oocyte retrieval; cyclical estradiol (E(2)) and progesterone (P(4)) production; restoration of serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels to reproductive age range. RESULT(S) Both patients were menopausal immediately after oophorectomy. The first patient developed a dominant follicle 10 weeks after transplantation, and her gonadotropin levels decreased to nonmenopausal levels. Percutaneous aspiration of ovarian follicles yielded a metaphase I (M-I) oocyte that was matured to metaphase II (M-II). The first patients graft was functional for at least 21 months. In the second patient, ovarian follicle development was detected 6 months after transplantation, and periodic menstruation occurred thereafter. Spontaneous ovulation was confirmed by a midluteal increase in her P(4) levels. Menstruation and follicle development continued for more than 2 years after the transplant. CONCLUSION(S) Heterotopic transplantation of ovarian tissue to the forearm is a simple and promising technique to restore ovarian function in women who become menopausal due to chemotherapy, surgery, or radiation.

Random anti-Müllerian hormone (AMH) is a predictor of ovarian response in women with elevated baseline early follicular follicle-stimulating hormone levels

OBJECTIVE To investigate the utility of random anti-Müllerian hormone (AMH) in assessing ovarian response among women with diminished ovarian reserve (DOR) diagnosed by elevated early follicular-phase FSH levels. DESIGN Retrospective study. SETTING Academic and academically affiliated assisted reproductive technology (ART) programs. PATIENT(S) Seventy-three women undergoing ART with elevated early follicular FSH levels. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Number of oocytes retrieved during ART cycle, number of day 3 embryos, and cycle cancellation and clinical pregnancy rates. RESULT(S) Random AMH levels were strongly correlated with the number of oocytes retrieved during an ART cycle among women with elevated FSH (r=0.55). Women with elevated FSH who had a random serum AMH level of 0.6 ng/mL or higher had twice the number of oocytes retrieved (11±1.3 vs. 5.6±0.6), a greater number of day 3 embryos (5.7±0.9 vs. 3±0.5), and approximately a third of the cycle cancellation rate (14% vs. 41%) compared with women with a random serum AMH below 0.6 ng/mL. The clinical pregnancy rate was also higher among women with a random serum AMH≥0.6 ng/mL (28% vs. 14%), however, the difference was not statistically significant. CONCLUSION(S) A random serum AMH level is useful in setting expectations for ART prognosis in women with elevated early follicular-phase serum FSH levels.

Leptin suppresses anti-Mullerian hormone gene expression through the JAK2/STAT3 pathway in luteinized granulosa cells of women undergoing IVF

STUDY QUESTION Do the adipocytokines, leptin and adiponectin affect the granulosa cell expression of anti-Mullerian hormone (AMH) and its receptor (AMHR-II)? SUMMARY ANSWER Leptin suppresses AMH mRNA levels in human luteinized granulosa cells through the JAK2/STAT3 pathway, while adiponectin has no such effect. WHAT IS KNOWN ALREADY AMH is one of the most reliable markers of ovarian reserve. Serum AMH levels decline with obesity. Obesity is associated with elevated leptin and reduced adiponectin levels. STUDY DESIGN, SIZE AND DURATION This prospective study included 60 infertile women undergoing fresh IVF and ICSI cycles utilizing autologous oocytes at Montefiores Institute for Reproductive Medicine and Health between July 2010 and April 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS Follicular fluid was collected from small (SFs; <14 mm) and large follicles (LFs; ≥14 mm) from 38 participants. Total RNA was extracted separately from mural and cumulus granulosa cells and mRNA levels were measured by RT-PCR. In an additional group of participants (N = 22), primary cumulus and mural granulosa cells (pooled SFs and LFs) were cultured in media alone or with addition of either leptin (N = 7), adiponectin (N = 8) or JAK2/STAT3 inhibitor + leptin (N = 7), and AMH and AMHR-II mRNA levels measured. Levels of AMH, leptin and adiponectin protein were measured in follicular fluid. MAIN RESULTS AND THE ROLE OF CHANCE AMH and AMHR-II mRNA and follicular fluid AMH protein levels were inversely correlated with age. AMH mRNA expression was six times higher in cumulus compared with mural granulosa cells in SFs (P< 0.05) and eight times higher in cumulus compared with mural granulosa cells in LFs (P < 0.001). In follicular fluid, leptin protein level positively correlated (r = 0.7, P = 0.03), while adiponectin protein level inversely correlated (r = -0.46, P = 0.02) with BMI. Leptin treatment suppressed AMH and AMHR-II mRNA in both cumulus and mural granulosa cells (all P < 0.05). In the presence of JAK2/STAT3 inhibitor, leptin treatment did not alter AMH but continued to suppress AMHR-II mRNA in cumulus cells (P = 0.02). Adiponectin treatment did not alter AMH or AMHR-II mRNA levels. LIMITATIONS, REASONS FOR CAUTION This study included a luteinized granulosa cell model as these cells were collected from women who were hyperstimulated with gonadotrophins. The results obtained may not fully extrapolate to non-luteinized granulosa cells. WIDER IMPLICATIONS OF THE FINDINGS Leptin may program abnormal AMH signaling, thereby resulting in ovarian dysfunction. This study opens a new perspective for understanding the low ovarian reserve seen in obese women and provides new insights into potential mechanisms that explain the lower AMH seen in obese women. Whether our findings explain the worse response to ovulation induction observed in obese women needs to be further elucidated.

Anxiety correlates to decreased blood and prefrontal cortex IGF-1 levels in streptozotocin induced diabetes

It is well known that diabetes mellitus may cause neuropsychiatric disorders such as anxiety disorders. Diabetes may also cause reduced IGF-1 (insulin like growth factor-1) levels in brain and blood. The purpose of the present study was to investigate the relationship between diabetes induced anxiety and IGF-1 levels in diabetic rats. The anxiety levels of rats were assessed 2 weeks after intraperitoneal injection of streptozotocin. Diabetic rats had higher levels of anxiety, as they spent more time in closed branches in elevated-plus-maze-test and less time in the center cells of open-field-arena. Prefrontal cortex (PFC) IGF-1 levels and neuron numbers were decreased and apoptosis was increased in diabetic rats. Blood IGF-1 levels decreased in a time dependent fashion following streptozotocin injection while blood corticosterone levels increased. They had higher malondialdehyde levels and lower superoxide dismutase enzyme activity. Oxidative stress may negatively affect blood and PFC tissue IGF-1 levels. Reduction in IGF-1 may cause PFC damage, which may eventually trigger anxiety in diabetic rats. Therapeutic strategies that increase blood and brain tissue IGF-1 levels may be promising to prevent psychiatric sequelae of diabetes mellitus.

Elevated body mass index is associated with lower serum anti-mullerian hormone levels in infertile women with diminished ovarian reserve but not with normal ovarian reserve

OBJECTIVE To investigate the association between elevated body mass index (BMI) and ovarian reserve. DESIGN Cross-sectional study. SETTING Academic institutions. PATIENT(S) Two hundred ninety women with infertility. Diminished ovarian reserve (DOR) was defined as day 3 FSH>10 IU/L. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Random serum antimullerian hormone (AMH) levels and number of oocytes retrieved during assisted reproductive technology cycle. RESULT(S) Increasing BMI was associated with lower random serum AMH levels in infertile women with DOR but not in women with normal ovarian reserve (NOR). Among women with DOR, mean random serum AMH levels were 33% lower in overweight and obese women compared with women with normal weight. The same association was not true for women with NOR. Out of 290 women evaluated for infertility, 109 women underwent a controlled ovarian hyperstimulation (COH)-IVF cycle. Women with elevated BMI and DOR had lower number of oocytes retrieved compared with women with normal BMI and DOR (6.4±4.3 vs. 9.4±6), an association that was not observed among women with NOR. CONCLUSION(S) Overweight and obese women with DOR as defined by high day 3 serum FSH levels have lower serum AMH levels and number of oocytes retrieved compared with nonobese women with DOR.

The c-Jun N-terminal kinase (JNK) functions upstream of Aurora B to promote entry into mitosis

Mitogen-activated protein kinases (MAPKs) are components of signaling cascades regulated by environmental stimuli. In addition to participating in the stress response, the MAPKs c-Jun N-terminal Kinases JNK1 and JNK2 regulate the proliferation of normal and neoplastic cells. JNKs contribute to these processes largely by phosphorylating c-Jun and thus contributing to the activation of the AP-1 complex. We here report that JNKs control entry into mitosis. We have observed that JNK activity and phosphorylation of c-Jun become elevated during the G2/M transition of the cell cycle in immortalized fibroblasts and ovarian granulosa cells. Pharmacological inhibition of JNK causes a profound cell cycle arrest at the G2/M transition in both cell types. This effect is specific as it occurs with two distinct small molecule compounds. Inactivation of JNK prior to mitosis prevents expression of Aurora B and phosphorylation of Histone-H3 at Ser 10. Silencing of JNK1 and 2 causes a similar effect, whereas overexpression of JNK1 and 2 causes the opposite effect. Inhibition of JNK delays activation of cdc-2 and prevents downregulation of Cyclin B1. We conclude that JNK signaling promotes entry into mitosis by promoting expression of Aurora B and thereby phosphorylation of Histone-H3.

Maternal treadmill exercise during pregnancy decreases anxiety and increases prefrontal cortex VEGF and BDNF levels of rat pups in early and late periods of life

In a previous study we demonstrated that, regular aerobic exercise during pregnancy decreased maternal deprivation induced anxiety. The purpose of this study is to investigate whether the positive effects of maternal exercise on the male and female offsprings early and late period of life. Half of the test subjects in each group were evaluated when they were 26 days old, and the other half were evaluated when they were 4 months old. The anxiety levels of maternally exercised groups were less than the control groups in both sexes and in both prepubertal and adult periods. The locomotor activity more increased in females. The prefrontal VEGF and BDNF levels were greater for both age groups and sexes in the maternally exercised group compared to control group. Moreover, there was a strong positive correlations between prefrontal cortex BDNF levels and results of open field tests; and VEGF levels and results of elevated plus maze tests. There was no difference in serum corticosterone levels between groups. These results indicate that maternal exercise during pregnancy may protect the pups from anxiety in early and late periods of life, and affects the prefrontal cortex positively.

Implications of blood type for ovarian reserve

BACKGROUND We explored the relevance of blood type to ovarian reserve, as reflected by early follicular phase FSH levels. METHODS For this cross-sectional observational study, early follicular phase serum levels of FSH (mIU/ml) and estradiol (E2, pg/ml), and information on blood type (A, B, AB and O) and patient age were procured for female patients, ≤ 45 years age (n= 544), who were undergoing fertility evaluation at one of two tertiary care facilities. Serum FSH > 10 mIU/ml was taken to reflect diminished ovarian reserve (DOR). Data distribution for FSH and age was analyzed and non-parametric tests used for comparisons across blood groups. Multivariable logistic regression analyses determined the relationship between elevated FSH and respective blood types after adjusting for age and study site. RESULTS Prevalence of blood types according to order of frequency was: O (45%), A (35%), B (16%) and AB (5%). After adjusting for age and study site, patients with blood type O were twice as likely to exhibit FSH > 10 mIU/ml compared with those with A or AB blood types [odds ratio (OR) 2.36; 95% confidence interval (CI) 1.27-4.41; P= 0.007], and three times as likely to manifest FSH > 12 m IU/ml (OR 3.48, 95% CI 1.46-7.32, P= 0.004). The B blood group antigen failed to exhibit any relationship with ovarian reserve as reflected by baseline FSH (P> 0.05). CONCLUSIONS The A blood group antigen appears to be protective for ovarian reserve, whereas blood type O appears to be associated with DOR, in a relationship that is independent of advancing age. Further studies are needed to establish causality and identify the underlying mechanisms for the association.