Ozgur Oktem

Understanding follicle growth in vivo

Ovarian reserve is determined by the number of primordial follicles in the ovary. Quiescent primordial follicles are activated for growth and pass through stages of development before they reach the antral stage. Then a cohort of antral follicles is recruited for further growth, dominance and ovulation under the cyclic stimulation of gonadotrophins. What triggers the initiation of growth in primordial follicles has remained a mystery for decades. However, recent studies on mutant mouse models have shown that primordial follicles are maintained in a dormant state by the actions of various inhibitory molecules to preserve the follicle pool, such as the transcription factor Foxo3a, PTEN (phosphotase and tensin homolog deleted on chromosome 10) and Tsc-1 (tumour suppressor tuberous schlerosis complex). Mice with deletions of these oocyte-specific genes exhibit premature activation of dormant primordial follicles, and all primordial follicles become depleted in early adulthood, causing premature ovarian failure. Other oocyte and somatic cell-derived growth factors are also involved in the early, gonadotrophin-independent phase of follicle growth via autocrine and paracrine interactions. Interestingly, some of these factors also play critical roles at later stages of follicle growth, such as the process of selecting the dominant follicle, by modifying the response of the follicles to gonadotrophins and inhibiting premature luteinization. Therefore, a thorough understanding of the molecular aspects of folliculogenesis is of paramount importance in the context of translational medicine and future clinical applications in human reproduction.

A Novel Ovarian Xenografting Model to Characterize the Impact of Chemotherapy Agents on Human Primordial Follicle Reserve

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.

Clinical outcome of intracytoplasmic injection of spermatozoa morphologically selected under high magnification: a prospective randomized study

Recent evidence shows that the selection of spermatozoa based on the analysis of morphology under high magnification (×6000) may have a positive impact on embryo development in cases with severe male factor infertility and/or previous implantation failures. The objective of this prospective randomized study was to compare the clinical outcome of 87 intracytoplasmic morphologically selected sperm injection (IMSI) cycles with 81 conventional intracytoplasmic sperm injection (ICSI) cycles in an unselected infertile population. IMSI did not provide a significant improvement in the clinical outcome compared with ICSI although there were trends for higher implantation (28.9% versus 19.5%), clinical pregnancy (54.0% versus 44.4%) and live birth rates (43.7% versus 38.3%) in the IMSI group. However, severe male factor patients benefited from the IMSI procedure as shown by significantly higher implantation rates compared with their counterparts in the ICSI group (29.6% versus 15.2%, P=0.01). These results suggest that IMSI may improve IVF success rates in a selected group of patients with male factor infertility. New technological developments enable the real time examination of motile spermatozoa with an inverted light microscope equipped with high-power differential interference contrast optics, enhanced by digital imaging. High magnification (over ×6000) provides the identification of spermatozoa with a normal nucleus and nuclear content. Intracytoplasmic injection of spermatozoa selected according to fine nuclear morphology under high magnification may improve the clinical outcome in cases with severe male factor infertility.

The ovary: anatomy and function throughout human life.

A prerequisite to the understanding of the ovarian diseases and infertility is a thorough understanding of normal embryology and physiology of the ovary. Therefore, the objective of this review article is to provide brief and updated information on the molecular basis of the events that control gonadal development, germ cell formation, folliculogenesis, and ovulation.

The Role of Extracellular Matrix and Activin-A in In Vitro Growth and Survival of Murine Preantral Follicles

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.

Removal of unilateral endometriomas is associated with immediate and sustained reduction in ovarian reserve.

Endometrioma surgery by stripping the cyst capsule has been associated with a reduction in ovarian reserve. It is still not clear whether the inflicted damage is immediate, sustained over time or associated with the use of electrocautery, nor which marker is more accurately reflects the post-operative reduction in ovarian reserve. This observational study assessed the damage inflicted by endometrioma removal with anti-Müllerian hormone (AMH) concentration and antral follicle count (AFC) pre and post-operatively. Twenty-five women with unilateral endometrioma underwent laparoscopic stripping of the endometrioma cyst capsule. There was a significant decrease both in AMH concentration (24%) and in AFC (11%) 1 month following surgery (P<0.01). At 6months post-operatively, the respective values were 24% and 15% less than preoperatively. AMH concentration and AFC showed no correlation with the use of bipolar electrocautery during surgery. Primordial follicles embedded adjacent to the cyst capsule were found in 61.5% of the specimens. Endometrioma surgery by stripping of the cyst capsule is associated with a significant reduction in ovarian reserve. The reduction is immediate and sustained over time. AMH appears to be a better indicator for post-operative quantification of the ovarian reserve.

Fertility preservation medicine: a new field in the care of young cancer survivors.

Treatment modalities for numerous oncological and non‐oncological conditions result in gonadal insufficiency and infertility. Furthermore, pelvic‐abdominal radiation may result in uterine damage resulting in poor reproductive outcomes such as preterm birth, low birth weight, and spontaneous abortion in adult survivors of childhood cancers. In response to the recognition of the impact of cancer treatments on fertility, several fertility preservation techniques have been developed. In prepubertal children, fertility preservation options are usually limited to ovarian cryopreservation because of sexual immaturity, but oocyte freezing can be performed in adolescent children. Two prospective randomized studies showed no benefit of gonadal suppression with GnRH analogs to preserve gonadal function and thus this treatment should not be recommended. For adult survivors of childhood cancer who experienced reproductive failure, third party reproduction techniques are highly successful. Pediatr Blood Cancer 2009;53:267–273.

Fertility preservation for breast cancer patients.

Breast cancer is the most common neoplasm in women and accounts for 26% (182,460) of all new cancer cases among women. With the use of screening mammography and advancement in other diagnostic modalities, many cases of breast cancer now can be diagnosed and treated at early stages of the disease. Unfortunately, adjuvant chemotherapy regimens commonly used in the treatment of breast cancer may cause premature ovarian failure due to their cytotoxic effects on the germ cells in the ovary. Therefore preservation of fertility in breast cancer survivors at reproductive age has become an important quality of life issue. Fertility preservation is a recently emerged field of reproductive medicine that may help protect the reproductive capability of the cancer survivors and allow them to have children in the future. Embryo freezing is the most established fertility preservation strategy. But conventional ovarian stimulation protocols are contraindicated in breast cancer patients because of the rise of estrogen and its metabolites to supraphysiological levels. Recently developed ovarian stimulation protocols with aromatase inhibitor letrozole and tamoxifen appear to provide a safe stimulation with endogenous estrogen levels comparable with those achieved in the natural cycle. Oocyte freezing can be considered in single women and in those who do not wish donor sperm. Ovarian tissue freezing could also be an option in breast cancer patients who do not wish or have a time for an in vitro fertilization cycle, which requires 10 to 14 days of ovarian stimulation.

Vitrified human ovaries have fewer primordial follicles and produce less antimüllerian hormone than slow-frozen ovaries

Slow-freezing and vitrification methods of human ovarian tissue cryopreservation were compared in terms of primordial follicle count and in vitro antimüllerian hormone (AMH) and estradiol production. Compared with fresh and slow-frozen ovaries, vitrified ovaries contained statistically significantly fewer primordial follicles and produced statistically significantly less AMH in vitro. Estradiol production from slow-frozen and vitrified ovaries was similar but statistically significantly lower than from fresh cultured strips.

Regeneration of Oocytes After Chemotherapy: Connecting the Evidence From Mouse to Human

In this issue, Dr Tilly’s laboratory presents another challenge to a half century– old dogma that ovarian reserve, made up of primordial follicles, cannot be replenished after birth. Their earlier work indicated that egg manufacturing continued into adult life in mice and that germ stem cells could have originated from the bone marrow; this work was met with much skepticism. It has been known for some time that bone marrow transplantation (BMT) and hematopoietic stem-cell transplantation (HSCT) are associated with extremely high rates of ovarian failure and infertility as a result of the preconditioning chemotherapy and radiotherapy. However, there are numerous reports of spontaneous pregnancies, even years after BMT or HSCT. Building on those observations and in response to various criticisms of their earlier work, Lee at al have tested the question of how BMTs can restore fertility in a rodent model. They found, to their surprise, that the donor-derived eggs from BMTs were not responsible for preservation of fertility in the recipients. All pups that were born after BMT were of recipient origin, even though a small fraction of immature oocytes of donor origin was found in recipient ovaries by cell tracking. The current concept that the ovary has a static ovarian reserve is entirely at odds with the germ cell dynamics in its counterpart, the testis. The testis contains a sizeable number of germ stem cells and continuously generates new gametes, even into old age. Although sperm numbers may diminish slightly at the extreme end of aging, recent research in mice suggested that this is mainly a result of aging of the niche rather than a defect in germ cells. The message that is given by Lee at al is that the ovary is more like the testis, at least when exposed to chemotherapy. It is not a static organ and continues to produce new germ cells into adult life. Speculatively, perhaps chemotherapy does not simply take away a number of eggs from a fixed ovarian reserve but interferes with the ability of the ovary to regenerate oocytes. If the mice born after BMT had the genetic background of the recipient, how did that procedure restore fertility? The fact that BMTs help maintain fertility despite the reduction in the size of follicle population after chemotherapy suggests that the transplantation may restore the lost germ stem cells, which then enables the ovary to maintain its reserve. Lee et al cleverly pointed out that the BMT treatment might have repaired chemotherapy-induced damage to the niche or the ovarian stroma. Indeed, our recent research showed that human ovarian samples taken from women who were previously exposed to various chemotherapy agents produce less estrogen than controls, indicating that chemotherapy may alter ovarian stromal function. In addition, there are clinical examples that suggest a role for ovarian niche in recovery from chemotherapy-induced ovarian failure. In a recent report, we described a Hodgkin’s lymphoma survivor who underwent subcutaneous ovarian transplantation with previously frozen autologous ovarian tissue. Before the transplantation, she was menopausal for 2.5 years as a consequence of an HSCT. Given the spontaneous recovery of ovarian function in the remaining menopausal ovary and recurrent spontaneous pregnancies concurrent with follicle development in the grafted ovary, we speculated that autologous ovarian transplantations may provide the necessary stromal oocyte recruitment/regeneration signals to the ovary that might have been damaged by previous chemotherapy. There could also be additional explanations for the better maintenance of fertility after BMTs. Because the animals that were administered sublethal doses of chemotherapy followed by BMT had lower mortality than the controls, the improved fertility might simply have been a result of improvement of the overall quality of life by that procedure. It would have been interesting to see whether mice treated with BMT better maintained their ovarian cyclicity compared with controls. Unfortunately, vaginal smears were not obtained in the study by Lee et al. If a higher fraction of animals continued to cycle in the BMT group, this would explain the higher pregnancy rates. The authors also observed that BMT had less benefit if mating was delayed for 2 months. Although they interpreted this as evidence for potential interaction between the hormonal milieu of pregnancy and BMT treatment in favor of future fertility, this could have also been a result of an accelerated loss of follicles during the months after chemotherapy. Previous work has suggested that primordial follicles that were exposed to chemotherapy and that seem to be morphologically normal under light microscopy may have ultrastructural damage and are eventually cleared from the ovary. In support of the interpretation of Lee et al, fertility was more severely impaired if BMT was also delayed for 2 months. Thus, it seems that, if BMT is to have a beneficial effect on fertility preservation, it should be performed as soon as the chemotherapy agents are cleared from the systemic circulation. The study did not address the mechanism of fertility protection by BMT, but the authors ruled out the possibility of an JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 25 NUMBER 22 AUGUST 1 2007