The success of in-vitro fertilization (IVF) treatment depends on adequate follicle recruitment. Controlled ovarian hyperstimulation (COH) is the gonadotrophin induced stimulation of the ovaries for purposes of IVF treatment, which specifically aims at inducing ongoing multiple follicle development rather than a single dominant follicle in ovulatory women.
Poor ovarian response to gonadotropin stimulation for IVF is not uncommon and is a predictor for low pregnancy rates (2%-4%).1,2 The incidence of poor response to assisted reproductive techniques (ART) is estimated to be 9%-24%.3 The definition of poor response to COH in IVF treatment varies widely based on one or a combination of the following factors: age (more than 40 years); an elevated follicle-stimulating hormone (FSH) on days 2 or 3 of the menstrual cycle, or previous cycle cancellation due to insufficient ovarian response; variable numbers of mature follicles observed on ultrasound (ranging from less than two to less than five), maximal oestradiol levels during COH (ranging from 100 to 660 pg/ml), number of mature oocytes retrieved (less than three to less than six), minimal cumulative dose, or days of gonadotropin stimulation required in a prior cycle.4,5
How to improve the clinical pregnancy rates of these special patients was one of the tough problems for IVF. We applied modified super-long down-regulation protocol to these patients in our reproductive center and, retrospectively, summarized the successful pregnancy rate.
The study involved 51 patients who responded poorly to high dose gonadotropin treatment in their previous cycles at our reproductive center, between April 2010 and February 2012. The pregnant patients in the previous cycle, who miscarried or asked for another baby would receive the subsequent cycle.
Poor ovarian response to gonadotropin stimulation for IVF at our center was defined as: few oocytes (≤5 oocytes, diameter ≥14 mm), and low peak estrogen (E2) level (<1650 pg/ml) until the day human chorionic gonadotrophin (hCG) was given; or high gonadotropin requirement (≥40-50 bottles, 75 U/bottle; ≥300 U/d, and ≥15 days) during routine long protocol, nonpregnancy or miscarriage.
The previous cycle (group A) received routine long protocol: gonadotrophin releasing hormone agonist (GnRHa) (short-acting triptorelin) 0.1 mg was injected hypodermically every other day at mid-luteal phase, the same dose was administered until the day hCG was given. Follicle stimulating hormone was used on day 5 of the subsequent menstruation.
The subsequent cycle (group B) received modified super-long down-regulation protocol: GnRHa (long-acting triptorelin) 1.25 mg was injected intramuscularly once on day 2, three weeks later, when E2≥20 pg/ml, follicle stimulating hormone was used. GnRHa (short-acting triptorelin) 0.05 mg was administered every day when at least one of the dominant follicles was 10-11 mm in diameter until the day hCG was given.
When at least one follicle had reached a diameter larger than 18 mm, recombinant human chorionic gonadotropin alfa (5000-10 000 U) was injected to promote the maturation of the oocytes. Oocytes were retrived after 34-36 hours.
Oocyte retrieved and insemination
Oocyte retrieval was performed by ultrasound-guided follicle aspiration under balanced anesthesia. Oocyte-cumulus complexes were incubated in G-Fert Plus medium. The cumulus oophorus and the corona radiata were subsequently removed from the oocytes by a short incubation in hyaluronidase solution followed by mechanical removal using glass pipettes. Oocytes were evaluated for their maturation state. In vitro fertilization/intra-cytoplasmic sperm injection (IVF/ICSI) was carried out using standard protocols. Embryos (1-3) at 4-8 cells were transferred. Clinical pregnancy was confined by elevated serum hCG level and pregnancy sac identified by ultrasonography.
Frequency of oocyte fertilization and pregnancy rate were compared for group A and B. Differences between the two groups were assessed by using Student's t test, and χ2 test where appropriate. All analyses were performed using the SPSS 11.0 package (SPSS Inc., USA).
The patients' average age was (36.64±3.85) years. The mean duration of ovarian stimulation cycles of the group A patients was longer than that of the group B patients. Levels of D3 FSH were higher (P <0.05) in group A than in group B (Table 1). The total dose of FSH was significantly lower in the subsequent cycle. The peak value of serum estradiol at hCG day was lower in group A, as compared with group B. Notably, both the number of Metaphase II oocytes and the rates of cleavage, were significantly higher in group B than in group A (P <0.001) (Table 1). The 49 patients in group B reached embryo transfer stage, while 46 patients in group A reached this stage. Patients in group B received significantly more embryos per transfer as compared with group A (P <0.001). More pregnancies and more clinical pregnancies with fetal heart activity were achieved in group B (Table 2).
The successful end-point of IVF treatment is for a woman to give live birth. This outcome is based on various factors including adequate number of retrieved eggs, which are obtained using various treatment protocols. Failure to recruit adequate follicles, from which the eggs are retrieved, is called a “poor response”.
The main cause of poor responses may include: (1) premature ovarian failure, (2) ovarian dysfunction, such as operation on an ovary, endometriosis,6 ovarian tuberculosis, ovaries ruined due to smoking, or impacted blood supply to the ovaries, (3) antibody to gonadotropins in patients, (4) heredity,7 and (5) agnogenic women: whose basal FSH and E2 level were normal, but poor response in COH. Various treatment protocols targeted at these women have been proposed, aiming to increase their ovarian response. Women who respond poorly to ovulation induction in the context of ART (poor responders) present a challenging and frustrating problem. Poor responders often have their treatment cycle cancelled because of presumed poor outcome in terms of clinical pregnancy.8 This can cause emotional distress for the couple as well as increase the financial burden on the service provider, couple, or both.
Various treatment protocols have been proposed targeting at this particular cohort of women, undergo IVF treatment after a poor response in a previous treatment cycle, aiming at increasing ovarian response to COH. These protocols can be reviewed as follows: (1) GnRH antagonist or GnRH agonist protocol;9–11 (2) “flare up” protocol (mini-dose GnRH-ag, macro-dose FSH, gonadotrophin releasing hormone-antagonist (GnRH-ant), HCG), or a modified natural cycle IVF (GnRH-ant, HCG);12–14 (3) adjuvant therapy such as use of oral contraceptive pills (COCP),15 GH,16 dehydroepiandrosterone (DHEA),17 androgens,18,19 progestins, L-arginine, etc.
The strategy using GnRH agonist/antagonist is to suppress pituitary gonadotrophin secretion and then to stimulate the follicular growth with gonadotrophins, thus facilitating cycle control. A long protocol of GnRHa to prevent premature LH surges has been the standard treatment for ovarian stimulation in assisted reproduction. In the long treatment protocols (with GnRHa started either in the mid-luteal phase or in the early follicular phase of the preceding cycle) gonadotrophin administration is delayed until pituitary desensitization has been achieved, which usually takes two to three weeks.20
In our trial, the previous cycle (group A) received routine long treatment protocol. The subsequent cycle (group B) received modified super-long down-regulation protocol. The total dose of gonadotropins used and the number of metaphase II oocytes recovered were improved significantly in group B. In group A, the addition of GnRHa for luteal suppression before ovarian stimulation for IVF does not improve IVF outcomes in poor responders.
Moreover, peak serum estradial concentration achieved at hCG day was higher in group B as compared with group A. We speculate that more follicles in the cohort were rescued by modified super-long down-regulation protocol and additionally higher levels of estradial were produced in group B. Higher concentrations of estradiol in pre-ovulatory follicular fluid predict a higher chance of pregnancy.21 Improved pregnancy rate in poor responders was likely due to inhibition of the abnormal internal environment following modified super-long down-regulation protocol management. Modified super-long down-regulation protocol appears to be a better method to be employed for poor responders.
In conclusion, this comparative trial shows that poor responder women undergoing repeated assisted reproduction treatment using modified super-long down-regulation protocol achieve more oocytes leading to higher fertilization rate, compared to women receiving routine long protocol. Our study also showed that clinical pregnancy rate was significantly increased. The limited number in our study might cause bias and a further large scale investigation is necessary.
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