Oocyte cryopreservation has wide clinical implications especially for women who have no partner or stand to lose ovarian function due to aging [
1]. Following embryo cryopreservation, oocyte cryopreservation is the second most commonly used method of fertility preservation for medical indications [
1]. In January 2013, the American Society for Reproductive Medicine declared that the technique of oocyte cryopreservation (egg freezing) is no longer experimental [
2]. In young women, there is good evidence that fertilization and pregnancy rates are similar to IVF/ICSI with fresh oocytes when vitrified/warmed oocytes are used as part of IVF/ICSI. Although data are limited, no increase in chromosomal abnormalities, birth defects, and developmental deficits has been reported in the offspring born from cryopreserved oocytes when compared to pregnancies from conventional IVF/ICSI and the general population.
Over the past decade, vitrification has been developed as an alternative to slow-freeze [
3,
4]. Most data in women suggest that post-thaw survival rates of vitrified oocytes are much higher than oocytes that underwent slow-freeze [
3,
4]. Vitrification protocols use high initial concentrations of cryoprotectant and ultra-rapid cooling to solidify the cell into a glass-like state in order to prevent the formation of ice crystals. Vitrification is currently being applied to the cryopreservation of embryos, oocytes, and ovarian tissue [
2,
5]. Embryo cryopreservation and oocyte cryopreservation in young women are well-established techniques among IVF centers [
6]. However, data pertaining to autologous oocyte cryopreservation by vitrification in older women, especially those older than 40, remain scarce.
Minimal and mild ovarian stimulation IVF usually refers to the use of low-dose gonadotropins with or without a sequential administration of clomiphene citrate or letrozole [
7]. Typically, minimal/mild ovarian stimulation usually refers to gentle stimulation protocols that yield a maximum of five to six oocytes [
7]. It has been suggested that the relatively small number of oocytes obtained after gentle ovarian stimulation may represent the best of the cohort in a given cycle [
8]. However, data pertaining to the outcome of autologous oocyte freezing, in particular live birth, in older women using minimal/mild ovarian stimulation IVF is understudied. Thus, the purpose of this manuscript was to report the outcome of frozen oocytes by vitrification in women aged 40 or older who underwent minimal/mild stimulation IVF.
Materials and methods
This is a retrospective chart review study that involved 158 women aged 40 or older who underwent minimal/mild ovarian stimulation IVF at a single fertility center (New Hope Fertility Center, New York). Reasons for autologous oocyte cryopreservation included: women who had no male partner, women who desired to preserve their future ability to have children because they wanted to delay childbearing, women who had fear of losing all oocytes due to aging, women with a family history of early menopause fearing that their oocytes would be depleted at an early age. All patients were counseled that the efficacy and the success (i.e., achieving a live birth) of autologous oocyte freezing is understudied in women aged over 40. They were also informed that the probability of getting pregnant with frozen oocyte could be low after the age of 40 although specific percentages could not be quoted. The studay was approved by the Institutional Review Board of New York Downtown Hospital (IRB approval reference number: JZ-09-08) [
9].
After oral contraceptive pill pre-treatment for approximately 3 weeks and adequate suppression, minimal/mild ovarian stimulation was started with an extended regimen (from cycle day 3 until the day before triggering) of clomiphene citrate (50 mg/day orally) or letrozole (2.5 mg/day) in conjunction with low dose of gonadotropin injections (75 IU/day and increased as needed to 150 IU/day) (Bravelle and/or Menopur, Ferring, Parsippany, NJ; Follistim, Merck, White House Station, NJ; or Gonal F, EMD Serono, Rockland, MA) starting on cycle day 4 to 7 depending on the response. Ovarian response was monitored using serial transvaginal ultrasound and serum blood measurement for estradiol, progesterone and luteinizing hormone. No hypothalamic-pituitary ovulation suppression using GnRH agonist or antagonist was used in this protocol. The final maturation of oocytes was induced by a nasal GnRH agonist (Synarel nasal spray 2 mg/mL, Pfizer, New York, NY) when the lead follicle is >18 mm. Retrieved oocytes were checked for maturity and mature oocytes were cryopreserved by vitrification.
Oocytes were vitrified using the Kitazato commercial kit (Dibimed Biomedical Supply S.L., Valencia, Spain) as previously descibed [
10‐
12]. The oocytes were aspirated with a minimal volume of HEPES culture media then placed in a micro-droplet containing the Basic Solution. The oocytes were allowed to acclimate to the new solution for approximately 10–30 s then placed in a drops of Equilibration Solution containing 7.5 % (vol/vol) ethylene glycol (EG) + 7.5 % dimethyl sulfoxide (DMSO) at room temperature for 15 min. After 6 min, the oocytes were transferred into a Vitrification Solution drop containing 15 % EG + 15 % DMSO + 0.5 M sucrose for a total of 50 s before being loaded into a Cryotop (fine and thin strip) that is quickly plunged into liquid nitrogen.
Oocyte thawing and fertilization by ICSI was performed for all couple when using either the male partner or donor sperm. While all semen parameters are important during an ICSI cycle, motility is the most important because it is an indication of living sperm. For this study all sperm injected into oocytes were motile. When choosing motile sperm for injection, basic head, neck and tail morphology were considered, and in all cases the best sperm available were chosen over any dysmorphic sperm in the samples. The embryos were cultured in a single global total medium (LifeGlobal Group LLC) that contains proteins, salts, amino acids, buffer (NaHCO3), glucose, pyruvate, lactate and antibiotics (gentamicin). All embryos were placed in incubators containing 5 % CO2 that results in a physiologic pH of approximately 7.30.
A single embryo was transferred in a subsequent artificially prepared cycle with oral estradiol (Estrace, Actavis Pharma, Inc, Parsippany, NJ) [
13]. All patients started oral estradiol at 4 mg/d orally for 10 days starting on the third day of menstruation. The dose of Estrace was subsequently increased to 6–8 mg/d in cases where serum estradiol level was less than 200 pg/mL and/or when ultrasound images showed an endometrial thickness of less than 7 mm. Oral estradiol treatment was then continued for 7–14 days until the endometrial thickness was more than 7 mm. Progesterone vaginal insert (prometrium) 200 mg three time daily was administered to support the luteal phase and oral estradiol was continued along with progesterone. Embryo transfer was performed on the 5th day of progesterone supplementation for cleavage stage embryos and on the 7th day of progesterone supplementation for blastocyst embryos. All embryo transfers were performed under in ultrasound guidance to ensure correct placement, which is 1–2 cm from the uterine fundus.
Data were presented as mean ± standard error of the mean. The main outcome was live birth per embryo transfer. Pearson correlation was performed to evaluate the association between age and clinical parameters such as day 3 FSH, peak serum estradiol level, number of oocytes retrieved, and number of mature metaphase II oocytes. Pearson correlation was also performed to evaluate the association between BMI and the number of oocytes retrieved and mature metaphase II oocytes. Statistical significance was declared if the two-sided p-value was ≤ 0.05. Statistical analyses were performed using statistical software GraphPad Prism six (GraphPad Software, Inc. La Jolla, CA, USA).