Faculty Bibliography

OBJECTIVE:To understand the basis of the phenotypic variations of 46, XX girls with mutations in the gene for Steroidogenic Acute Regulatory (StAR) Protein. The patients with mutation in both the alleles of the StAR gene result in deficiency of all the steroidal hormones and severe adrenal insufficiency. The majority of the 46, XX females spontaneously undergo puberty but the underlying defect ultimately leads to the development of ovarian cysts and premature menopause. The mechanism of the lesion in the ovary remains to be understood completely. DESIGN/METHODS:We compiled the description of the clinical information and biochemical data of patients with StAR mutation from published manuscripts. These articles were collected from the NCBI website (www.pubmed.org) and from the subsequent reference searches of retrieved articles. The data of the 46,XX patients with proven StAR mutation were included for the review. RESULTS:The majority of StAR 46,XX females developed irregular menses and ovarian cysts. The ovarian cyst enlargement progressively led to torsion and presented as a life-threatening emergency. The fertility of 46,XX StAR patients is severely compromised as ultimately premature menopause ensued. CONCLUSIONS:Early hormonal replacement is warranted to prevent the progressive ovarian cyst formation. Newer techniques to preserve the fertility of these patients can be implied early in the pubertal developmental process if patients desire pregnancy.

BACKGROUND:Various chemotherapy agents, especially of the alkylating category, have been associated with premature ovarian failure but there is no quantitative evidence of chemotherapy-induced ovarian damage in humans. The aim was to quantify the impact of chemotherapy on primordial follicle reserve and stromal function in human ovary with a prospective controlled quantitative histologic and in vitro study. METHODS:Samples from 26 patients who were undergoing ovarian tissue cryopreservation for fertility preservation were assessed histologically and for in vitro estradiol production. Of the 26 patients, 10 had previously received chemotherapy whereas 16 had not (control). There were 17 age-matched patients. Primordial follicle counts and in vitro estrogen production were compared between age-matched control and chemotherapy patients as well as between those who did and did not receive alkylating agents. RESULTS:Patients who received chemotherapy had significantly lower primordial follicle counts than control patients (5.4 +/- 1.32 vs 9.6 +/- 2.2, P = .05). Furthermore patients treated with alkylating regimens had significantly lower primordial follicle counts compared with those who received nonalkylating agents (2.9 +/- 1.1 vs 7.9 +/- 1.6, P < .05) and with those who did not receive any chemotherapy (2.9 +/- 1.1 vs 9.6 +/- 2.2, P < .05). In vitro, ovarian cortical pieces from individuals who were previously exposed to chemotherapy (chemotherapy group) produced significantly less estradiol compared with those who were not (control group). In the chemotherapy group, there was no difference in in vitro estradiol production between those who received alkylating agents and those who did not. CONCLUSIONS:This is the first quantitative evidence of the impact of chemotherapy on ovarian infrastructure, and shows that, whereas alkylating agents can significantly reduce ovarian reserve, both alkylating and nonalkylating regimens may affect ovarian stromal function.

Many chemotherapeutic agents, especially of the alkylating family, alter fertility in premenopausal females. However, it is not practically possible to quantify and characterize the impact of cancer drugs on ovarian reserve in a clinical setting. Thus, our specific aim was to develop a xenograft model to characterize the in vivo impact of chemotherapy agents on human ovary. Ovarian pieces from 24 weeks old abortuses were xenografted s.c. to severe combined immunodeficient mice (n = 52). Animals received either a single dose of 200 mg/kg of cyclophosphamide or the vehicle. Grafts were recovered from the control and treated mice 12 to 72 h after the cyclophosphamide injection and serially sectioned for primordial follicle counts. Apoptosis was assessed with terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) assay. Platelet/endothelial cell adhesion molecule 1 (PECAM-1) staining, as well as intra-vital fluorescein-conjugated lectin and Evans blue labeling were done to assess microvasculature by confocal microscopy. Although there was 12% reduction in primordial follicle density by 12 h following treatment (P > 0.05), the follicle loss increased significantly at 24 h (53%, P < 0.01) and peaked at 48 h (93%, P < 0.0001). TUNEL staining peaked at 12 h, earlier than the diminishment in follicle numbers, and decreased thereafter. Xenograft vascularization pattern was similar to non-xenografted tissue, indicating appropriate in vivo drug delivery. The impact of cyclophosphamide on primordial follicle reserve in our human ovarian xenograft model is consistent with the clinical gonadotoxicity of this drug. Human ovarian xenografting is a promising model to characterize the gonadotoxic effects of current and emerging cancer drugs without a need for lengthy clinical studies.

OBJECTIVE:To reduce estrogen exposure in women with endometrial cancer undergoing in vitro fertilization using an aromatase inhibitor. DESIGN/METHODS:Prospective case series. SETTING/METHODS:Academic center for reproductive medicine. PATIENT(S)/METHODS:Endometrial carcinoma patients presenting for fertility preservation or fresh embryo transfer to gestational carrier. INTERVENTION(S)/METHODS:Four patients with endometroid carcinoma underwent five IVF cycles for immediate or delayed embryo transfer to gestational carriers before or after staging and definitive surgery. To prevent surge in E(2) levels, letrozole was started 2 days before gonadotropin administration and then given concomitantly. Embryos were either cryopreserved for fertility preservation or transferred freshly to a surrogate. MAIN OUTCOME MEASURE(S)/METHODS:Peak E(2) level during stimulation, pregnancy in a gestational carrier. RESULT(S)/RESULTS:Peak E(2) level during stimulation was 386.67 +/- 102.93 pg/mL. A mean of 7 +/- 2.85 oocytes were retrieved, resulting in 4.8 +/- 1.76 embryos per cycle. In one patient, two embryos were transferred to a surrogate, resulting in a triplet pregnancy. The delivery occurred at 31 weeks and the infants did not show any congenital or developmental abnormalities. Three patients had their embryos cryopreserved for future use. CONCLUSION(S)/CONCLUSIONS:The use of letrozole and gonadotropins is associated with lower E(2) levels compared with standard stimulation cycles in endometrial carcinoma patients. Combination of this approach with surrogacy may enable these young women to preserve their fertility.

PURPOSE OF REVIEW/OBJECTIVE:This review focuses on the current options for fertility preservation in young women facing the risk of premature ovarian failure and infertility as a sequel to the treatment of gynecologic cancer. RECENT FINDINGS/RESULTS:There is a wide range of options to preserve fertility. Embryo freezing is the most established method and the success rate of in-vitro fertilization using frozen-thawed embryos now approaches that of using fresh embryos. Success rates with oocyte cryopreservation are on the rise and begin to approach that of embryo freezing. Exposure to high levels of estrogen during ovarian stimulation can be minimized by utilizing aromatase inhibitors in women with estrogen-sensitive cancers undergoing embryo or oocyte cryopreservation. Ovarian-tissue cryopreservation is used to preserve fertility in children and in patients with time restraints; however, the number of reported cases is very small. Likewise, in-vitro maturation and xenografting are experimental and their potential to preserve fertility remains to be determined. SUMMARY/CONCLUSIONS:A number of fertility-preservation techniques have been developed and many others are in the experimental stages. Consistent with recent ASCO clinical guidelines, all young individuals with gynecologic cancer should be counseled about the available fertility preservation techniques.

Every year, an increasing number of women with malignant and nonmalignant diseases is successfully treated with cytotoxic chemotherapy and/or radiotherapy. Many of these patients suffer from infertility and gonadal failure as a result of these treatments. At present, these patients may resort to assisted-reproduction techniques to protect their future childbearing potential before the implementation of cytotoxic therapy. While embryo cryopreservation is an established technology, oocyte and ovarian tissue freezing techniques are still investigational. Nevertheless both of these techniques have resulted in live births. Apart from assisted-reproduction techniques, it has been extensively debated whether administration of gonadotropin-releasing hormone (GnRH) analogues in conjunction with chemotherapy can protect ovarian reserve against cytotoxic insult. In this manuscript, we debate the rationale for the effectiveness of GnRH analogue coadministration in preservation of fertility by reviewing the literature, and provide preliminary data to support our views.

CONTEXT/BACKGROUND:Breast cancer patients undergoing controlled ovarian hyperstimulation (COH) for embryo or oocyte cryopreservation should be induced by the method that leads to the least increase in estradiol (E(2)) levels. OBJECTIVE:The aim of the study was to determine the potency of anastrozole to suppress serum E(2) levels in breast cancer patients undergoing COH. DESIGN AND SETTING/METHODS:A prospective sequential cohort study was conducted in an academic center for reproductive medicine between May 2003 and November 2005 for letrozole and between December 2005 and April 2006 for anastrozole. PATIENTS/METHODS:Breast cancer patients presenting for fertility preservation participated in the study. INTERVENTION/METHODS:COH using FSH and letrozole (n = 47) or anastrozole (n = 7) was followed by oocyte retrieval and embryo cryopreservation. MAIN OUTCOME MEASURES/METHODS:Serum E(2) levels, area under the curve for E(2), and outcomes of COH cycles were measured. RESULTS:There were no significant differences between the two groups regarding length of stimulation, total gonadotropin dose, number of follicles larger than 17 mm, and the lead follicle size on human chorionic gonadotropin (hCG) day and number of embryos cryopreserved. The mean E(2) levels on the day of hCG and post-hCG days were higher in the anastrozole group compared to the letrozole group (1325.89 +/- 833.17 and 2515.07 +/- 1368.52 vs. 427.78 +/- 278.24 and 714.38 +/- 440.83 pg.d/ml; P < or = 0.01), respectively, even when anastrozole dose was increased up to 10 mg/d. The mean area under the curve was significantly higher in the anastrozole group compared to the letrozole group (4402.93 +/- 1526.7 vs. 1287.48 +/- 732.17 pg.d/ml; P <0.004). CONCLUSIONS:Breast cancer patients who underwent ovarian stimulation with anastrozole had a significantly higher exposure to E(2) than those who were stimulated with letrozole.

Extracellular matrix plays a key role in cell growth, survival, and differentiation in a wide array of tissue types through integrin-mediated signaling pathways and its interaction with growth factors. This study investigates the role of extracellular matrix and its interaction with activin-A on in vitro growth and survival of mouse preantral follicles. Preantral follicles isolated from 14-day-old immature mouse ovaries were cultured either 3 dimensionally using basement membrane matrix (growth factor - reduced matrigel) or 2 dimensionally on cover slips coated with a single component of extracellular matrix (fibronectin, collagen, or laminin), on polylysine (negative control), or in standard culture plates in a serum-free culture medium with or without activin-A for 7 days. Follicles cultured in matrigel maintained well their 3-dimensional structure compared to those cultured conventionally. This observation was confirmed by analyzing 3-dimensional images of follicles cultured in matrigel and standard culture plate using confocal microscopy. Furthermore, follicles displayed higher growth and survival rates and exhibited antral space formation as early as day 5 of culture when activin-A was added to matrigel; in contrast, the addition of activin-A had no effect on the growth and survival of follicles cultured on individual extracellular matrix components after 7 days of culture. These data may suggest that 3-dimensional culture with extracellular matrix and activin-A provides a better milieu for in vitro growth and survival of preantral follicles in immature mice.

CONTEXT/BACKGROUND:Diploid/triploid mosaicism (mixoploidy) is a rare chromosomal abnormality characterized by mental and growth retardation, hypotonia, and dysmorphic features such as facial asymmetry, low-set ears, and syndactyly. All 46,XX/69,XXY cases fall into three phenotypic groups: male with testicular development, ovotestis disorder of sex development (DSD), or undervirilized male DSD. All phenotypic females with diploid/triploid mosaic reported so far had 46,XX/69,XXX karyotype. PATIENT/METHODS:We report an 8-year-old girl conceived after in vitro fertilization-intracytoplasmic sperm injection with normal internal/external genital and ovarian development despite 46,XX/69,XXY mosaicism and normal expression of sex-determining region of Y chromosome (SRY) in her gonads. INTERVENTION/METHODS:Because of the increased risk of gonadoblastoma resulting from Y chromosome mosaicism, her ovaries were removed by laparoscopy. Ovarian tissue was analyzed histologically as well as by fluorescence in situ hybridization, PCR, and RT-PCR amplification to determine the localization of Y chromosome and expression of SRY and DAX1 mRNA. Methylation-specific PCR was used to assess the inactivation pattern of X chromosomes. RESULTS:By laparoscopy, internal female genital anatomy appeared to be normal. Cytogenetic and molecular methods confirmed the presence of intact and functionally active Y chromosome in the ovary. Strikingly, histological assessment of the gonads showed normal ovarian architecture with abundant primordial follicles despite the presence of the Y chromosome in ovarian follicles and the expression of SRY mRNA in gonadal tissue. CONCLUSION/CONCLUSIONS:This case illustrates that normal ovarian development is possible in the presence of Y chromosome in ovarian follicles and despite the expression of SRY in ovarian tissue. Furthermore, this is the first documented case of mixoploidy after in vitro fertilization-intracytoplasmic sperm injection and the only phenotypic female with 46,XX/69,XXY karyotype.