“A case report on a new strategy of fertility preservation in women with borderline ovarian malignancy”

Jack Y.J. Huang, William M. Buckett, Lucy Gilbert, Seang Lin Tan, Ri-Cheng Chian ⁎ Department of Obstetrics and Gynecology, McGill University Health Center, McGill University, Montreal, Quebec, Canada H3A 1A1

Received 22 December 2006 Available online 26 March 2007

Abstract

Background. We report a novel fertility preservation strategy in a woman with borderline ovarian tumors involving retrieval of immature oocytes, in vitro maturation (IVM) and subsequent cryopreservation.

Case. A 43-year-old woman underwent laparotomy for cystic ovarian masses on day 18 of her menstrual cycle. A diagnosis of bilateral borderline ovarian tumors was made following histological frozen section analysis. Left salpingo-oophorectomy, right ovarian cystectomy, omentectomy and lymph node sampling were performed. All visible follicles on the surface of the removed ovary were aspirated. Four immature oocytes were retrieved and underwent IVM. Three oocytes matured after 48 h and were cryopreserved.

Conclusion. Immature oocytes can be successfully isolated from the oophorectomy specimen regardless of the day of menstrual cycle, matured in vitro and cryopreserved, providing a possible strategy for fertility preservation in this group of women.

© 2007 Elsevier Inc. All rights reserved.

Keywords: Borderline; Low malignant potential; Ovarian cancer; Malignancy; Fertility preservation; Cryopreservation; Oocyte; In vitro maturation; Vitrification

Introduction

Borderline or low malignant potential ovarian tumors account for 10% to 15% of all ovarian epithelial tumors. Standard management of borderline tumors is similar to that of malignant ovarian tumors and includes surgical staging, total hysterectomy (TH), bilateral salpingo-oophorectomy (BSO), omentectomy, lymph node sampling, peritoneal sampling and cytoreductive surgery. Given the fact that a third of borderline ovarian tumors are diagnosed in patients aged less than 40 years and 75% of these tumors present as FIGO stage 1 at the time of diagnosis, conservative surgery defined as surgery with complete staging but where the uterus and at least a part of one ovary are preserved has been advocated [1]. However, conservative surgery is not suitable for patients who previously had unilateral oophorectomy, those with bilateral bulky tumors or advanced stage disease. This group, as well as patients who are at high risk of recurrence in the conserved ovary, would benefit from additional strategies for fertility preservation.

We report a novel possibility for fertility preservation in a woman with borderline ovarian tumors involving retrieval of immature oocytes from the oophorectomy specimen followed by in vitro maturation (IVM) of these oocytes and subsequent cryopreservation by vitrification.

Case report

A 43-year-old woman (gravida 1, para 0, aborta 1) underwent an explorative laparotomy for bilateral ovarian masses and a CA125 of 47 U/ml. Prior to the surgery, the patient was counseled about the risk of malignancy, but wished to preserve her fertility if possible.

Intraoperative findings included a 8 × 8 × 6 cm right ovarian cyst and two cysts, measuring 4 × 3 × 3 cm and 2 × 2 × 2 cm respectively, in left ovary. The uterus appeared normal with no evidence of other intra-abdominal pathology. Peritoneal wash- ing and bilateral ovarian cystectomy were performed. A frozen section histological assessment of the excised ovarian cysts suggested bilateral serous borderline ovarian tumors. A left salpingo-oophorectomy, right ovarian cystectomy and biopsy, omentectomy, pelvic lymph node sampling and palpation for aortic lymphadenopathy were performed on day 18 of her menstrual cycle.

The oophorectomy specimen was suspended in cold (4 °C) Leibovitz L-15 medium (Gibco Invitrogen, CA, USA) and transferred to the embryology laboratory. All visible follicles were aspirated with an 18-gauge needle that was attached to a 10 ml syringe. The aspirates were flushed in Oocyte Washing Medium (Sage/Cooper Surgical, CT, USA) and searched for cumulus–oocyte complexes under a dissecting microscope. Four immature germinal vesicle (GV) stage oocytes were isolated from the excised left ovary.

For in vitro maturation, the GV oocytes were incubated in an Organ Tissue Culture Dish (60 × 15 mm; Falcon) containing 1.0 ml of IVM-Medium (Cooper Surgical, CT, USA) supplemented with a final concentration of 75 mIU/ml of follicle- stimulating hormone (FSH) and luteinizing hormone (LH) at 37 °C in an atmosphere of 5% CO2 in air with high humidity [2]. After maturation in culture for 24 h, the cumulus cells were denuded using finely drawn glass pipettes after 1 min of exposure to 0.1% hyaluronidase solution (Cook, Brisbane, Australia). At 24 h post-IVM, two oocytes matured to metaphase-II stage (MII). Following culture for an additional 24 h, one remaining immature oocytes reached MII stage.

The in vitro matured oocytes were cryopreserved using the McGill Cryoleaf (MediCult, Jyllinge, Denmark) and vitrification method as previously described [3]. For the vitrification procedure, the oocytes were suspended in equilibration medium containing 7.5% (v/v) ethylene glycol (EG) + 7.5% (v/v) 1,2-propanediol (PROH) for 5 min at room temperature and then transferred to the vitrification medium containing 15% (v/v) EG + 15% (v/v) PROH + 0.5 M sucrose at room temperature for 45–60 s. They were loaded on the McGill Cryoleaf and then plunged immediately into liquid nitrogen for storage.

The final pathology confirmed the diagnosis of bilateral serous cystic, micropapillary type and borderline ovarian tumors. There was no evidence of neoplastic implants or metastatic tumors in the peritoneal washing, omentum and lymph nodes.

At the six-month post-operative follow up, the patient remained disease free and reported having normal menstrual cycles.

Discussion

It is now generally accepted that fertility sparing surgery is a safe and effective option in women of reproductive age with stage 1 borderline ovarian tumors who have not completed childbearing [1]. This case demonstrates that, in this group of patients, immature oocytes can be successfully isolated from the oophorectomy specimen, matured in vitro and vitrified, providing an additional possibility for fertility preservation. Even in stage 1 patients in whom the contralateral ovary is normal, and the affected ovary is removed to reduce the risk of recurrence, the retrieval of immature oocytes from the excised ovary for IVM and crypreservation is worth considering to improve fertility prospects in the future.

Interestingly, the immature oocyte retrieval was performed in the luteal phase on day 18 of her menstrual cycle, indicating that regardless the day of menstrual cycle immature oocytes can be collected and matured in vitro. It is possible that a higher number of oocytes may be retrieved if this procedure is performed during the follicular phase of the cycle prior to ovulation and in younger women.

Our strategy may also be beneficial to women of reproductive age with early invasive ovarian cancer or other types of gynecologic cancer who need to undergo sterilizing surgery. Following IVM, if a male partner is present, the matured oocytes may be inseminated by in vitro fertilization (IVF) and the resultant embryos may be cryopreserved. If the patient is single, matured oocytes can be cryopreserved by vitrification. In hysterectomized patients, embryos could be transferred to a surrogate.

This novel strategy of fertility preservation is a safer option in women who are diagnosed with borderline ovarian tumors than ovarian tissue cryopreservation. Borderline tumors arise in the epithelial cells of the ovarian cortex. Therefore, transplantation of the cryopreserved ovarian cortical tissues carries the risk of reimplanting neoplastic epithelial cells.

Unlike transvaginal ultrasound-guided follicle puncture and oocyte retrieval which may result in inadvertent rupture of cystic tumors and intraperitoneal spillage of tumor cells, in this case the oocyte retrieval from the oophorectomy specimen was performed under a sterilized fume hood in the clinical embryology laboratory. Moreover, prior to cryopreserving the oocytes, all the cells surrounding the oocytes were enzymatically removed. Therefore, we believe that the risk of transferring neoplastic cells to the oocytes and resultant embryos is unlikely.

Ovarian stimulation, retrieval of mature oocytes and IVF have been performed in patients with borderline ovarian tumors following conservative surgery, resulting in pregnancy and live births [4]. However, the safety of ovarian stimulation in a patient suspected of ovarian malignancy prior to disease excision remains unclear and controversial. One cycle of ovarian stimulation is equivalent to 2 years of normal menstrual cycles in term of the level of estrogen achieved [5]. In animal model studies, gonadotropins stimulated the proliferation of borderline ovarian tumor and epithelial ovarian cancer [6]. In fact, FSH binding receptors were identified in epithelial ovarian cancer cell lines and FSH was found to have a stimulatory effect on papillary serous ovarian cancer cell division. However, in a more recent in vitro study, estradiol and FSH were noted to have no effect on epithelial neoplastic cell proliferation. In a case report describing a patient with stage IIIc serous micropapillary LMP tumors, ovarian hyperstimulation resulted in malignant transformation and rapid peritoneal dissemination of tumor cells [7]. Therefore, based on the current findings from animal and clinical studies, ovarian stimulation treatment prior to disease excision should be offered only with caution.

Another consideration is that one ovarian stimulation treatment protocol entails a four- to six-week delay in order to undergo pituitary down-regulation followed by gonadotropin stimulation and oocyte retrieval if the standard long protocol is used. Even if a short protocol is performed, there would be a delay of at least 2 to 3 weeks. Therefore, immature oocyte retrieval followed by IVM and cryopreservation of these oocytes is a possible option in patients with time constraint and concerns regarding the long-term effect of gonadotropin stimulation.

IVM has become an effective treatment option for many infertile women, namely those with high antral follicle count, poor response IVF treatment, polycystic ovaries or polycystic ovary syndrome and those at risk of developing ovarian hyperstimulation syndrome [8]. In fact, more than 300 healthy infants have been born to date. In this case, a maturation rate of 75% was achieved following IVM, demonstrating that a high percentage of GV stage oocytes from unstimulated ovaries can reach MII stage using this IVM culture system.

Oocyte cryopreservation by vitrification is a promising new technique and appears to be more effective than the conventional slow-freezing and has been used to preserve fertility in cancer patients [9]. In clinical studies using conventional slow freezing/ rapid thawing method, the mean survival rate approximated 50% and varied markedly between the reported cases and series (23% to 89%) [10]. In the past 5 years, several groups have reported high survival rates (89.2% to 100%) and successful live births using vitrification methods. In fact, since the first report in 1999, vitrification of oocytes has resulted in more than 30 live births worldwide, including 9 from our department [3,10].

In conclusion, immature oocytes can be successfully isolated from the oophorectomy specimen regardless of the day of menstrual cycle, and undergo IVM and cryopreservation. Patients with ovarian tumors who wish to preserve their fertility may benefit from this additional and novel possibility for fertility preservation.

References

  1.  Cadron I, Amant F, Van Gorp T, Neven P, Leunen K, Vergote I. The management of borderline tumours of the ovary. Curr Opin Oncol 2006;18:488–93.
  2. Chian RC, Buckett WM ,Tulandi T, Tan SL .Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Hum Reprod 2000;15:165–70.
  3. Chian RC, Son WY, Huang JY, Cui SJ, Buckett WM, Tan SL. High survival rates and pregnancies of human oocytes following vitrification: preliminary report. Fertil Steril 2005;84:S36.
  4. Morice P. Borderline tumours of the ovary and fertility. Eur J Cancer 2006;42:149–58.
  5. Fishel S, Jackson P. Follicular stimulation for high tech pregnancies: are we playing it safe? BMJ 1989;299:309–11.
  6. Cramer DW, Welch WR. Determinants of ovarian cancer risk. II. Inferences regarding pathogenesis. J Natl Cancer Inst 1983;71:717–21.
  7. Attar E, Berkman S, Topuz S, Baysal B, Akhan S, Chambers JT .Evolutive peritoneal disease after conservative management and the use of infertility drugs in a patient with stage IIIC borderline micro-papillary serous carcinoma (MPSC) of the ovary: case report. Hum Reprod 2004; 19:1472–5.
  8. Chian RC, Lim JH, Tan SL. State of the art in in-vitro maturation. Curr Opin Obstet Gynecol 2004;16:211–9.
  9. Rao GD, Chian RC, Son WS, Gilbert L, Tan SL. Fertility preservation in women undergoing cancer treatment. Lancet 2004;363:1829–30.
  10. Huang J, Tan SL, Chian RC. Fertility preservation for female. J Reprod Contracep 2006;17:109–28.