Young age does not protect against the adverse effects of reduced ovarian reserve—an eight year study

Tarek El-Toukhy, Yacoub Khalaf,1, Roger Hart, Alison Taylor and Peter Braude

Assisted Conception Unit, 4th Floor, Thomas Guy House, Guy's and St Thomas' Hospital NHS Trust, St Thomas' Street, London SE1 9RT, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Ovarian reserve significantly influences IVF outcome. Low response to ovarian stimulation due to reduction of ovarian reserve is occasionally encountered in young women. The aim of this study was to evaluate the outcome of IVF treatment in young patients with reduced ovarian reserve. METHODS AND RESULTS: Between January 1993–2001, 762 consecutive patients satisfied the definition of reduced ovarian reserve (raised early follicular phase FSH or gonadotrophin stimulation cycles where three or fewer oocytes were retrieved after routine FSH stimulation) and were included in the study. They were classified into three age groups: young (<=30 years), intermediate (31–38 years) and older (>38 years). The three age groups were similar with respect to basal (day 3) serum FSH and estradiol concentrations, cause of infertility and number of previous treatment cycles. Implantation (13, 9.6 and 9.8%), clinical pregnancy (11.8, 10.2 and 10%) and live birth (7.4, 7.3 and 6.8%) rates were not significantly different in the three age groups respectively (P > 0.05). CONCLUSION: This study shows that younger patients with reduced ovarian reserve have a poor outcome of IVF treatment similar to their older counterparts. Such information may be helpful in counselling these patients who otherwise might anticipate an outcome related to their chronological age.

Key words: IVF outcome/poor ovarian reserve/poor responders/young age


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The decline of female fertility with advancing age is well documented (Spira, 1988Go; Maroulis, 1991Go). It has been reported that approximately one third of women who defer pregnancy until their late thirties and over one half of those >40 years of age will have difficulty conceiving (Virro and Shewuck, 1984Go; Alrayyes et al., 1997Go). Within the context of IVF, as a woman's age increases her response to stimulation and likelihood of a live birth decreases. This largely reflects an age-related decline in ovarian reserve. However, in some women the decline in ovarian function occurs earlier. This usually manifests with a raised serum FSH level in the early follicular phase (day 2–4) of the menstrual cycle (i.e. basal FSH) and/or an inadequate response to a standard gonadotrophin stimulation protocol (Lindheim et al., 1996Go; Faber et al., 1998Go; Surrey et al., 1998Go) and predicts a poor cycle outcome.

It has been suggested that this poor prognosis may not be applicable to young poor responders (<=30 years) who might be protected from the adverse effects of reduced ovarian reserve by virtue of their age (Hanoch et al., 1998Go) and, therefore, should expect a better outcome than their older peers. This study investigates the outcome of assisted conception in different age groups of women showing signs of poor ovarian reserve whilst undergoing ovarian stimulation for IVF and embryo transfer, in order to determine whether younger patients truly have a better prognosis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Data about patients undergoing IVF or ICSI in our unit are routinely collected prospectively and stored in a relational database (FileMaker Pro 4.1 for Macintosh, Inso Corporation, USA).

Study population
Between January 1993–2001, all patients who fulfilled our definition of poor ovarian reserve; i.e. a raised basal serum FSH level >10 IU/l, or an inadequate ovarian response in a previous IVF/ICSI treatment cycle resulting in the retrieval of three or less oocytes in response to a standard stimulation protocol of 150–300 IU of FSH daily, were included in the study. Enrolled patients underwent ovarian stimulation with our maximum dose of stimulation (450 IU of FSH daily) for a subsequent IVF/ICSI cycle(s). The study population had had basal estradiol concentrations recorded concomitantly with FSH measurements. Only the first cycle after the diagnosis of reduced ovarian reserve was made was included in the study to avoid including the same patient twice.

Treatment protocol
All patients underwent pituitary down-regulation using buserelin (Suprefact; Hoechst UK Ltd., Hounslow, Middlesex, UK) in the mid-luteal long protocol. FSH treatment was commenced following satisfactory pituitary suppression. Ovarian stimulation was achieved using a daily FSH dose of 450 IU of purified urinary FSH (Metrodin HP; Serono Laboratories Ltd, Welwyn Garden City, UK) or recombinant FSH (Gonal-F; Serono, or Puregon; Organon, Cambridge, UK). Ovarian response during stimulation was monitored by transvaginal ultrasound measurement of follicle size (Roest et al., 1995Go). HCG 10 000 IU (Profasi; Serono, or Pregnyl; Organon) was administered to induce oocyte maturation when at least three follicles reached a mean diameter of >=18 mm. Transvaginal follicular aspiration was carried out 34–36 h later using an ultrasound scanner with a 6.5 MHz probe (EUB 525; Hitachi, Tokyo, Japan). Each visible and accessible follicle, regardless of size, was aspirated. Cycles were cancelled before HCG administration when fewer than three follicles had reached >=14 mm after 12 days of ovarian stimulation.

IVF
Oocytes were observed under an inverted microscope for evidence of fertilization 16–18 h after insemination. Fertilization was considered normal when two clearly distinct pronuclei were present. Cleavage-stage embryos were assigned grades on the second day after oocyte retrieval as described previously (Steer et al., 1992Go).

ICSI
After removal of cumulus/corona cells, the oocytes were examined for their nuclear maturity using an inverted microscope. ICSI was carried out on oocytes that had extruded the first polar body. Following sperm injection, the oocytes were transferred to 100 µl droplets of IVF medium under oil and incubated overnight at 37°C under 5% CO2. On the following day (16 h after injection) the oocytes were checked for survival and fertilization.

Embryo transfer
Depending on availability, between one and three cleavage-stage embryos were transferred to the uterus 48–72 h after insemination using an Edwards-Wallace embryo transfer catheter (Sims Portex Ltd., Hythe, Kent, UK) replacing the best grade embryos. All patients who underwent embryo transfer received supplemental progesterone pessaries 400 mg daily throughout the luteal phase.

Cycle outcome
Pregnancy was confirmed by a positive urine test for HCG ~14 days after embryo transfer. A clinical pregnancy was defined as the presence of a fetal heart beat on ultrasound scanning between 2 and 3 weeks after the positive pregnancy test. All pregnancies reported in this series were followed to delivery.

Because the present work involved no therapeutic interventions nor any change to our routine IVF protocols, we did not require additional approval from our institutional ethics committee. However, written informed consent was obtained from each couple upon entering our IVF programme.

Statistical analysis
Patients were classified according to age into three groups: young (<=30 years) (range 25–30 years), intermediate (31–38 years) and old (>38 years) (range 39–43 years). Statistical analysis was performed with use of analysis of variance (ANOVA) for different variables including the nature and mean number of ampoules of FSH used, duration of stimulation, number of oocytes retrieved, proportion of patients requiring ICSI and oocyte fertilization rate. Implantation, pregnancy and live birth rates per cycle were analysed. Associated clinical variables were compared with Student's-t test, {chi}2 or Fisher's exact test where appropriate. Statview software package (Abacus Concepts Ltd., Berkeley, CA, USA) was used for statistical analysis. Statistical significance was set at P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During the study period, 762 consecutive patients with poor ovarian reserve were included and treated. They were divided into three age groups (<=30, 31–38 and >38 years). A comparable proportion of each group was included because of raised basal FSH level (58, 45 and 44% respectively, P = 0.09, not significant). Demographic and stimulation data according to age group are shown in Table IGo. Apart from age and duration of infertility, there were no significant differences between the three groups with regard to number of previous IVF/ICSI treatment cycles, mean basal FSH and estradiol serum concentrations before treatment, nature of FSH used (whether urinary-derived or recombinant), duration of gonadotrophin stimulation required before oocyte retrieval and number of FSH ampoules required to effect ovarian stimulation. The primary causes of infertility were similar in the three age groups (Table IIGo). All groups showed comparable stimulation and cycle outcome with no statistically significant difference detected in relation to cancellation rate prior to oocyte retrieval, mean number of oocytes retrieved and fertilized normally and the proportion of patients requiring ICSI. There was no inter-group difference in oocyte fertilization rate, proportion of cycles with no oocytes collected or with total fertilization failure, mean number of embryos available on the day of embryo transfer and mean number of embryos replaced per patient.


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Table I. Patients' demographic and stimulation characteristics
 

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Table II. Causes of infertility in the three age groups. Values are percentages of the total number of patients in each group
 
The overall clinical pregnancy and live birth rates in the study population (n = 762) was 10.2 and 7% respectively; significantly lower than the corresponding average rates in our entire IVF/ICSI programme during the same study period (24 and 20% respectively; P < 0.01). The pregnancy, implantation and live birth rates per cycle started and per embryo transfer were not significantly different in the three age groups (Table IIIGo). Additionally, in the subset of the study population who conceived with treatment, the miscarriage rate was similarly high in the three groups (50% in those patients <=30 years, 52% in patients 31–38 years and 39% in the group >38 years; P = 0.7, not significant).


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Table III. Cycle outcome in the three age groupsa
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Female age is traditionally recognized as the major determinant of the outcome of assisted conception (Check et al., 1994Go; Templeton et al., 1996Go). Although features of reduced ovarian reserve are encountered in older women as a natural physiological trend toward menopause, some young women may occasionally exhibit similar features. The purpose of the present study was to determine the impact of age on IVF/ICSI outcome in those women already demonstrated to have reduced ovarian reserve. To our knowledge, this is the largest study to date comparing IVF/ICSI results in different age groups of patients having diminished ovarian reserve.

We used basal FSH level and the number of oocytes retrieved to define ovarian reserve. Basal FSH is an important signal of ovarian reserve and reflects the quality of the follicular apparatus as a whole (Muasher et al., 1988Go; Toner et al., 1991Go), while the number of oocytes retrieved is a more direct indicator of ovarian reserve and responsiveness (Silber et al., 1997Go). Both parameters have been reported to correlate well with the outcome of assisted conception (Scott et al., 1989Go; Toner et al., 1991Go; Dor et al., 1992Go; Khalaf et al., 1995Go; Meniru and Craft, 1997Go) and are in line with criteria mentioned by other authors (Akman et al., 2000Go). Comparable proportions of the three groups had high basal FSH levels or produced three or fewer oocytes.

Since an elevated basal estradiol level can suppress a potentially elevated FSH level (Smotrich et al., 1995Go), care was taken to have concurrent basal estradiol and FSH levels measured to prevent the introdution of bias in the parameters for cohort inclusion. Basal estradiol levels were compared in the different age groups and yielded comparable results (Table IGo), attesting to the fact that all three groups generally had a similar degree of poor ovarian reserve. In addition, the different age groups were shown to have comparable primary causes of infertility, a factor reported to have some effect on IVF outcome (Tanbo et al., 1995Go; Templeton et al., 1996Go).

The number of oocytes recovered following stimulation with gonadotrophins is known to be related to the total oocyte density found in the ovary (Lass et al., 1997Go). All three age groups were stimulated with the same daily dose of FSH (450 IU), responded by producing comparable mean number of oocytes and had a similar mean number of cycles with no oocytes retrieved. Thus, it seems reasonable to postulate that the patients included in the present study had comparable pools of recruitable follicles remaining in their ovaries with respect to number, sensitivity and responsiveness to gonadotrophin stimulation and therefore belong to one group prognostically, regardless of their age. Such postulation is supported by the fact that women having IVF treatment could be grouped into prognostic categories based on their response to a standardized stimulation regimen (Rosenwaks and Muasher, 1986Go). Additionally, and in agreement with a previous report (Out et al., 2000Go), the high FSH dose used in this study did not compensate for the decline in ovarian reserve observed in the younger age groups.

The present study demonstrates that IVF/ICSI outcome is not different for young poor responders <=30 years of age when compared with their older peers. The comparable mean number of retrieved oocytes and normal oocyte fertilization rate in the three age groups accord with previous studies (Oehninger et al., 1995Go; Silber et al., 1997Go; Biljan et al., 2000Go) showing no difference in fertilization rate between young and older poor responders, and confirm that these patients, irrespective of their age, share an inherent ovarian/oocyte problem referred to as `ovarian ageing'. In addition, embryo availability as well as the pregnancy, implantation and live-birth rates reported here (Table IIIGo) did not vary with age group. Such results strongly suggest that ovarian age is an independent variable critically affecting IVF outcome, while chronological age assumes a less important prognostic role once the diagnosis of diminished ovarian function has been made. This theory is supported by previous reports (Mosher and Pratt, 1991Go; Scott and Hofmann, 1995Go) which stressed that the age of onset of the decline in ovarian reserve is highly variable and therefore age alone could have a limited predictive value with respect to reproductive potential. In fact, basal FSH level, as a measure of ovarian reserve, has been shown to be a better predictor of a woman's chance to conceive than her chronological age (Scott et al., 1989Go; Toner et al., 1991Go).

Our study result contrasts with two previous studies which suggested that within poor responders, young patients have a significantly better cycle outcome than older patients (Roest et al., 1996Go; Hanoch et al., 1998Go). In those two studies, ovarian response was assessed either by adequacy of follicular recruitment (Roest et al., 1996Go) or by peak serum estradiol level measured on day of HCG injection (Hanoch et al., 1998Go). However, both studies assessed ovarian response retrospectively, included a relatively small number of cycles (227 and 143 respectively) and did not report live-birth or miscarriage rates. More importantly, patients included in those studies were stimulated with different doses of gonadotrophins depending on their age, thus potentially introducing bias in the group selection. It is possible that the inadequate response observed in the younger age group was due to the use of more modest stimulation protocols as previously suggested (Silber et al., 1997Go) and not necessarily secondary to diminished ovarian reserve. It can also be argued that in young poor responders, suboptimal follicular recruitment and/or estradiol secretion may not entirely reflect poor ovarian reserve or oocyte quality, but rather may reflect the relatively milder regimen of ovarian stimulation used for the younger group (Simon et al., 1998Go; Biljan et al., 2000Go). In the present study, the diminished ovarian reserve was confirmed prior to patient recruitment and all patients enrolled into the study subsequently received the same stimulation protocol and FSH dosage for the same duration and their ovarian response and cycle outcome were recorded prospectively.

There is considerable evidence that ovarian ageing is associated, not only with oocyte depletion, but also with a concomitant increase in the number of chromosomal abnormalities, particularly aneuploidy, affecting the remaining oocytes (Richardson and Nelson, 1990Go; Navot et al., 1991Go; Munné et al., 1995Go; Battaglia et al., 1996; Magli et al., 1998Go; Nasseri et al., 1999Go). Theoretically, oocytes from young patients suffering from the same pathological process of ovarian ageing as their older peers might also be susceptible to pre- or post-zygotic chromosome anomalies. The observed high frequency of miscarriage in our study population (47%) lends some credence to this theory. Because oocyte age is known to be the major factor in predicting the frequency of miscarriage (Lervan et al., 1991Go), the high miscarriage rate could be explained by the waning oocyte quality in all three age groups. This concurs with the study of Nikolettos and colleagues (2000) which reported a 50% miscarriage rate after IVF/ICSI treatment in 107 patients with reduced ovarian reserve (Nikolettos et al., 2000Go). A more recent study (Levi et al., 2001Go) reported a pregnancy loss rate of 71.4% in those who conceived among a group of 1034 infertility patients who had a raised basal FSH level. Nasseri et al. karyotyped products of conception from a group of women who underwent dilatation and curettage for miscarriage and found a significantly higher rate of fetal aneuploidy among women with diminished ovarian reserve (Nasseri et al., 1999Go). Unexplained recurrent miscarriage in various age groups has also been associated with reduced ovarian reserve presumably due to poorer quality oocytes with inherent chromosomal compromise (Trout and Seifer, 2000Go). Altered meiotic spindle formation, common among oocytes from older women, was implicated in reproductive loss in patients of all ages with diminished ovarian reserve (Battaglia et al., 1996).

While the mechanism of age-related decline in IVF success rates is believed to be due to a reduction in both quantity and quality of the oocytes, the cause of the accelerated decline in fertility in some young women is less clear. It has been shown (Faddy et al., 1992Go) that the rate of follicular attrition is not constant but rather follows a bi-exponential pattern, with the change in exponential rate being determined by the number of remaining oocytes rather than age. Therefore, differences in the number of oocytes at the beginning of reproductive life and in the rate of oocyte depletion thereafter determine the age at which ovarian reserve becomes seriously compromised. The authors (Faddy et al., 1992Go) concluded that the ovary measures biological time according to the number of oocytes remaining rather than chronologically. In this respect, our group has demonstrated (Lawson et al., 2001Go) that poor responders to gonadotrophin ovarian stimulation, regardless of age, are significantly more likely to be amenorrhoeic and to suffer from vasomotor symptoms requiring hormone replacement and reduced reproductive potential 5 years after their IVF treatment cycle compared with normoresponders. Whilst environmental and lifestyle factors such as smoking, body weight and socio-economic status can only partly explain this phenomenon (van Noord et al., 1997Go; Sneider et al., 1998Go), a recent study of singleton and twin sisters (de Bruin et al., 2001Go) emphasised the role that genetic factors play in the occurrence of early menopause and, consequently, early reproductive failure due to rapid decline in the size of the follicular store. Further studies addressing the same issue in young poor responders would be helpful.

IVF treatment is both expensive and stressful for patients. The expected chances of achieving a successful pregnancy are crucial in the decision as to whether an infertile couple decide to embark on IVF treatment. Thus, it is important to be able to counsel as accurately as possible about true prognosis. While chronological age is traditionally considered the major predictor of outcome, prognosis needs to be modified according to other factors; an important one of which is `ovarian age'. The present study concludes that young age does not protect against the adverse effects of reduced ovarian reserve and that it is appropriate to predict a poor outcome when young women exhibit features of diminished ovarian reserve. We emphasise that poor cycle outcome in young patients undergoing ovarian stimulation using high doses of gonadotrophins reflects an age-independent decline of ovarian reserve. The mechanism(s) of such an accelerated decline remain to be elucidated.


    Notes
 
1 E-mail: yakoub.khalaf{at}kcl.ac.uk Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on November 9, 2001; accepted on January 29, 2002.