Priming with a gonadotropin-releasing hormone agonist before immature oocyte retrieval may improve maturity of oocytes and outcome in in vitro maturation (IVM) cycle: a case report

The conception of in vitro maturation (IVM) of oocytes was first reported in 1935 [1]. Experiments were described in which ova taken from fallopian tubes at various intervals after fertile mating were cultured in vitro. A successful IVM cycle in humans using immature oocytes retrieved from antral follicles was reported later, followed by the first delivery from IVM of oocytes recovered from patients with untreated polycystic ovarian syndrome (PCOS) [2]. As early as 1994, PCOS patients were the first natural candidates for IVM [2]. Recently, the range of clinical applications of IVM has been emphasized as an additional opportunity for germ cell preservation in women suffering from cancer [3]. Novel radio- and chemotherapy treatments can significantly improve the prognosis of these patients but, unfortunately, have major effects on ovarian function, often leading to premature ovarian failure. Technically, immature oocytes collected from antral follicles in the absence of gonadotropin administration may be cryopreserved before or after maturation in vitro. Therefore, IVM may represent a suitable opportunity for recovery of oocytes intended for cryopreservation in cases where tumor estrogen sensitivity and/or urgency to commence therapy prevent a full controlled ovarian stimulation [3]. However, different studies have reported that implantation and pregnancy rates after IVM were lower than with traditional in vitro fertilization (IVF), and varied from 5 to 22% [4]. It is possible that in an IVM cycle, although nuclear maturation of metaphase II (MII) oocytes obtained after maturation in proper medium progresses normally, the cytoplasmic maturation is delayed, resulting in poorer embryonic development, implantation and pregnancy rates [5].

Human chorionic gonadotropin (hCG) has been the gold standard for ovulation induction as a surrogate for the mid-cycle luteinizing hormone (LH) surge. In recent years, the concept of using a gonadotropin-releasing hormone agonist (GnRH-a) for triggering ovulation was reintroduced among patients treated by an antagonist protocol for IVF. Interestingly, it was suggested that it may promote oocyte nuclear maturation, that is, resumption of meiosis and cumulus expansion [6]. It was recently suggested that triggering final oocyte maturation with GnRH-a versus hCG in IVM cycles among breast cancer patients undergoing fertility preservation could be beneficial, as evidenced by the number of mature oocytes and cryopreserved embryos [7]. Therefore, it seems that this case report may add to existing knowledge regarding the beneficial effect of GnRh-a as a triggering approach in consecutive IVM cycles performed for fertility preservation in patients with malignancies or uncommon indications.

A 38-year-old Caucasian housewife was referred to the IVF and Reproductive Genetics Center, Moscow, for fertility preservation. She and her family lived in a small nearby town with normal ecological conditions. There was no history of serious illness in her family. She used barrier contraception and had never been pregnant. She had never smoked or consumed alcohol.

She had regular, 25–28-day menstrual periods. Her past history revealed that she had undergone laparoscopy and unilateral right adnexectomy due to a large papillary mucinous cystadenoma, 10 cm in diameter. Ten years later, a new cyst, 4 cm in diameter, was diagnosed on the contralateral ovary. Laparotomy with partial ovarian resection was performed. Histological examination again revealed mucinous cystadenoma. She was otherwise healthy, height 166 cm and body mass index (BMI) 24.7.

In accordance with Russian legislation, ovarian stimulation in women with ovarian tumors (even small) is prohibited. Therefore, a natural cycle IVF was commenced, but no oocyte was retrieved.

In order to improve results and obtain oocytes without stimulating the ovaries, we decided to conduct an IVM cycle. A total of three successive IVM attempts were performed.

U/S monitoring was started on day 6 of the cycle. Endometrial thickness was 5 mm, and three antral follicles, 7.5 mm, 8.5 mm and 6 mm in diameter, were visualized on the left ovary. On day 8 of the cycle, the follicles were 8 mm, 9.5 mm and 7 mm, and triptorelin (Decapeptyl, Ferring GmbH, Kiel, Germany) 0.2 mg was administered subcutaneously to trigger oocyte maturation. Transvaginal U/S-guided follicle aspiration was performed 39 hours later using a 19G/17G single lumen needle (Swemed Sense, Vitrolife, Göteborg, Sweden) and a reduced aspiration pressure of 7.5 kPa. Follicular fluid was collected without flushing into 50 mL dishes with Oocyte Washing Medium (SAGE IVM media kit, Cooper Surgical, USA). A total of three oocyte-cumulus complexes (COCs) were obtained. All COCs were cultured in maturation medium (SAGE IVM media kit) supplemented with FSH+LH (Merional, IBSA Institut Biochimique SA, Switzerland) for a final concentration of 75 mIU/mL. Retrieved oocytes were stripped, found in the MII stage and fertilized by intracytoplasmic sperm injection (ICSI) 6 hours after aspiration as described previously [8]. All oocytes were fertilized 18 hours after ICSI. Two top-quality embryos (8 cells, grade 1) and one good-quality embryo (8 cells, grade 2) were vitrified 72 hours after fertilization. No luteal phase support was given to the patient.

Ten days after commencement of the menstrual cycle, endometrial thickness was 6.5 mm, and five antral follicles 10, 10.5, 8, 6 and 6 mm in diameter were demonstrated. Triptorelin 0.2 mg was injected subcutaneously. Ovum pickup was performed 38.5 hours later.

Six COCs were obtained, cultured in maturation media, stripped and fertilized by ICSI 4.5 hours after pickup. Normal fertilization occurred in five oocytes; the sixth developed one pronucleus. One top-quality embryo (10 cells, grade 1) was vitrified 72 hours after ICSI. Other embryos were cultured until day 6; two poor-quality blastocysts were obtained and discarded.

On the next menstrual cycle, U/S performed on day 10 revealed endometrial thickness of 7 mm and four antral follicles 10, 8, 6 and 5 mm in diameter. Transvaginal follicle aspiration was performed 39 hours after subcutaneous injection of 0.2 mg triptorelin as described above. Three COCs were obtained, cultured in maturation media for 5 hours, stripped, found to be in MII stage and fertilized by ICSI. Two developed 2 pronuclei (2PN) 18 hours after fertilization. Two good-quality embryos were vitrified on day 3. All IVM cycles were well tolerated, with no side effects.

In summary, 12 COCs were obtained following IVM of small antral follicle aspiration. All oocytes were found to be mature (MII) about 4.5 hours after incubation in maturation media. The fertilization rate after ICSI was 10/12 (83%) and cleavage rate was 10/12 (83%). A total of six good-quality embryos were vitrified on day 3. The patient recently underwent uneventful cystectomy ruling out malignancy. Frozen embryo transfer is planned.

We report here for the first time that repeated IVM cycles with GnRH-a triggering were successful and resulted in the collection of six high-quality embryos.

The emerging technology of IVM and oocyte retrieval has recently become an additional option for fertility preservation. This procedure can be done without hormonal stimulation and within a short time frame, as oocytes are collected during the follicular phase or even luteal phase treatment with a reasonable number of harvested oocytes [9]. Therefore, in cases of patients with malignancies, especially when hormone treatment is contraindicated, and in those who must start chemotherapy without delay or in unusual cases, IVM might be a preferred option to preserve fertility.

Oktay et al. [10] were the first to report the use of a GnRH analog as an oocyte maturation trigger for women with breast cancer undergoing IVF, and found that the number of oocytes retrieved, maturation and fertilization rates, and the number of 2PN embryos were significantly higher than with hCG trigger. Similar results were reported in breast cancer patients undergoing IVM cycles for urgent fertility preservation [8]. Recently, Dahan et al. [11] described GnRH-a triggering in a modified natural IVF cycle in a patient with PCOS. In this approach, follicles were stimulated with gonadotropins for 3 to 5 days when they were small, and ovulation was triggered with a GnRH-a when the largest follicles were 10–12 mm in diameter. Many immature oocytes were retrieved, matured in vitro and subsequently developed to form blastocysts that resulted in a live birth.

The rationale for triggering with a GnRH-a instead of hCG, mainly in an IVM cycle, is the induction of FSH surge comparable to the surge of the natural cycle. This surge may promote the formation of LH receptors on granulose cells, enhancing LH activity, inducing plasminogen activator activity that enhances dissociation of oocytes from the follicle wall (therefore more immature oocytes can be obtained), maintaining the opening of the gap junction between the cumulus cells and oocyte (enhancing oocyte maturation) [12, 13], promoting cumulus expansion [14] and possibly promoting cytoplasmic maturation thought to be delayed in IVM cycle triggering with hCG before oocyte retrieval. There is evidence that the release of LH and FSH after single administration of GnRH-a in an IVF cycle is able to complete the final stage of follicular maturation, resulting in retrieval of fertilizable oocytes, with normal embryo development and pregnancy [6]. Therefore, the combined action of GNRH-a, FSH and LH may have a beneficial effect on oocyte maturation, mainly in an IVM cycle.

In our three IVM cycles, 12 oocytes were obtained. Interestingly, all oocytes (100%) were found to be mature (MII) after 4.5–5 hours of culture in maturation media, in contrast to results in IVM cycles after hCG triggering, where only 15.6% were found to be mature up to 6 hours after retrieval, versus 64.9% that matured in vitro after 6–48 hours [15]. Indeed, from a semantic point of view, it could be suggested that in our case, the mature eggs obtained a short time after retrieval, which gave rise to high-grade embryos with possible high implantation potential, are not "really" IVM oocytes. However, the most important point is the result of obtaining six high-quality embryos that were vitrified.

To the best of our knowledge, this case report is one of the few reports in the literature of an unusual case involving GnRH-a priming before oocyte retrieval in an IVM protocol. Moreover, originally, we performed three successive cycles, accumulating high-quality embryos. Therefore, it seems that triggering ovulation with GnRH-a instead of hCG in an IVM cycle performed for fertility preservation or rare cases, as described herein, could be valuable in terms of obtaining higher-grade embryos, possibly due to more synchronous nuclear and cytoplasmic maturation.

The present report describes a new alternative for treating uncommon cases of infertility using consecutive repeated IVM cycles with GnRH-a triggering.