To analyze the association between oocyte quality and decreased ovarian reserve (DOR) markers in young women undergoing controlled ovarian stimulation (COS).
This retrospective study included 49 patients classified as having DOR based on anti-Müllerian hormone (AMH) levels, follicle-stimulating hormone (FSH) levels, or antral follicle counts (AFCs; <10). Images of all obtained oocytes were analyzed, and oocyte quality was classified according to maturity and morphology. The COS protocol utilized gonadotropin (FSH and/or human menopausal gonadotropin [hMG]) doses ranging from 150 to 300 IU/day. The Student’s
Women with DOR classified according to FSH level had more immature oocytes (
This study showed that DOR based on AMH levels, FSH levels, and AFC was associated with poorer quality oocytes in young women who underwent COS.
The age of a woman who wants to become pregnant is considered an important marker of ovarian reserve and a predictor of success in assisted reproductive technology. Thus, theoretically, younger women (<35 years of age) have a higher chance of becoming pregnant because they have more and higher quality oocytes [
Women with decreased ovarian reserve (DOR) are those of childbearing age with regular menstrual cycles and decreased response to controlled ovarian stimulation (COS) and/or lower fertility than other women of the same age [
The natural decrease in fertility and increased rates of miscarriage owing to aneuploidy that are associated with increasing age in women are established in the literature and have been suggested to result from the poor quality of remaining oocytes [
Morphological oocyte evaluation is simple and safe and provides high-fidelity oocyte quality parameters [
It is controversial whether variations in oocyte morphology occur due to ovarian hyperstimulation [
Few studies have correlated DOR with oocyte quality in young women and investigated whether this relationship impacts on success rates in assisted reproductive technology. Therefore, the objective of this study was to investigate whether an association between DOR (evaluated using three measurements: basal FSH levels, AMH levels, and AFCs) and oocyte quality according to morphology and maturity exists in young women. We also evaluated whether the gonadotropin dose used in COS was associated with an increased risk of oocyte alterations according to the ovarian reserve in these women.
This retrospective study analyzed data from patient records of infertile women who underwent COS for
Women aged between 18 and 35 years with a history of infertility and with or without previous attempts at assisted reproductive technology were included. The exclusion criteria were as follows: use of hormonal contraceptives or hormone replacement therapy during the 30 days prior to the assessment of ovarian reserve, history of pelvic radiotherapy or chemotherapy, oophorectomy and/or oophoroplasty, endocrinopathies (polycystic ovary syndrome, hyperprolactinemia, and hypopituitarism), autoimmune diseases, or genetic diseases identified by clinicians and confirmed by karyotyping. Patients with incomplete data in their medical records, such as those with missing examination results, were excluded.
Venous blood samples were collected from all included patients on the 2nd or 3rd day of their menstrual cycle to analyze basal FSH levels using chemiluminescence (IMMULITE 2000 XPi, Siemens Healthineers, Erlangen, Germany). AMH levels were measured using an enzyme-linked immunosorbent assay (AMH Gen II, Beckman Coulter, Inc., Brea, CA, USA).
Antral follicles were counted by the same observer using transvaginal ultrasonography. Follicles were considered antral when their size on transvaginal ultrasound was between 2 and 8 mm on the 2nd or 3rd day of menstruation. AFC was the sum of follicles found in both ovaries.
Patients were classified as having normal ovarian reserve (NOR) when the basal FSH levels were <10 mIU/mL, AMH levels were ≥1.2 ng/mL, or AFC was ≥10. Patients with FSH levels >10 mIU/mL, AMH levels <1.2 ng/mL, or AFC <10 were considered to have DOR.
The COS protocol utilized gonadotropins (FSH: follitropin beta, Puregon®, Organon, Jersey City, NJ, USA; follitropin alfa, Gonal-F, Merck Serono, Darmstadt, Germany; human urofollitropin, Fostimon®, UCB, Brussels, Belgium; and/ or human menopausal gonadotropin [hMG]: menotropin, Menopur®, Ferring Pharmaceuticals, Kiel, Germany) and was based on an individually tailored dose ranging from 150 to 300 IU/day according to age, ovarian reserve, and number of oocytes desired. FSH was replaced by hMG at the same dosage in all patients from the 6th day of ovarian stimulation. The combined doses of FSH and hMG administered over the entire stimulation period were considered the total dose. The gonadotrophin dose was fixed at the beginning and did not undergo modification during the COS cycle. Only one COS cycle was performed for each patient.
Based on the results of serial ultrasound examinations, ovarian blockage with a GnRH antagonist (ganirelix, Orgalutran ®, Organon, Ravensburg, Germany) was initiated when more than two follicles of ≥14 mm in size were observed. Urinary human chorionic gonadotropin [hCG] (5,000 IU; Choriomon®, UCB, Brussels, Belgium) or recombinant hCG (Ovidrel, Merck Serono, Darmstadt, Germany) was administered when three or more follicles of >18 mm were observed. Under patient sedation, oocytes were retrieved vaginally by ultrasound at 35–36 hours after the luteinizing hormone peak.
Archived images of all harvested oocytes were analyzed using an ECLIPSE Ti Series inverted microscope (Nikon, Tokyo, Japan) at ×200 magnification by the same embryologist. Oocyte quality was classified according to maturity and morphology (
Because sperm quality is an important factor for determining fertilization rate and embryonic development and the medical center where the treatments were performed is a reference for treating male-factor infertility, we chose not to assess oocyte fertilization rates and perinatal outcomes.
The student’s
Among 534 patients treated during the study period, 182 were excluded because they were older than 35 years, 259 did not meet the clinical criteria for inclusion, and 44 had incomplete data in their medical records. The main cause of infertility was severe male factor (34.7%), followed by ovulatory factor (14.3%). Forty-nine patients were included, and 348 oocyte images were analyzed. The mean patient age was 31.1±3.3 years and the mean body mass index was 23.2±2.5 kg/m2.
The distribution into groups according to patient NOR or DOR differed based on the measurements (
Patients with DOR defined by AMH levels or AFCs had higher basal FSH levels than those with NOR (
An association was found between AMH levels <1.2 ng/mL and the probability of having an immature oocyte (OR, 3.3; 95% CI, 1.2–8.8;
When DOR was defined according to the AFC, an increase in the risk of abnormally shaped oocytes was observed (OR, 2.9; 95% CI, 1.1–7.6;
No significant relationship was observed between the presence of alterations in the zona pellucida and the variables for ovarian reserve or between patient age and the applied oocyte quality parameters.
Our results showed that women up to 35 years of age who undergo COS exhibit poor oocyte quality, namely alterations in morphology and immaturity, when they present with DOR.
The higher number of immature and abnormal oocytes in women with DOR according to AMH levels, even if they had normal FSH levels, revealed that FSH alone may not be a good indicator of oocyte quality. Indeed, it has been suggested that basal FSH is a late marker of ovarian reserve, which corroborates the difference observed in our results [
As a result, AMH and AFC levels are good markers for predicting ovarian response to hormonal stimulation and pregnancy [
AMH and AFC levels are important predictors for the early identification of DOR before it reaches a critical level [
Our findings suggest that AMH is a strong marker of oocyte quality in young women who undergo COS as changes in the cytoplasm are considered to be the most significant factors affecting embryo outcome and implantation potential [
The results of the present analysis are corroborated by those of a previous prospective study that evaluated the rate of aneuploidy in groups defined according to ovarian reserve and included 327 patients who underwent
Our patients with NOR defined by AFCs had a greater number of mature oocytes (
Patients with oocytes having normal perivitelline space have been shown to have a higher rate of fertilization (60.3% when normal vs. 37.5% when increased) and better embryonic development [
No differences related to the total dose of FSH/hMG administered during ovarian stimulation were observed between any of the groups, and no increased risk of morphological alterations to oocytes was observed when this variable was considered. Therefore, it can be assumed that the quantity of medication administered to patients in this study did not have a significant impact on oocyte quality, which could have affected the analysis.
One limitation of our study was that a critical analysis of intra-observer reliability was not possible for AFCs as this was a retrospective study that only involved collecting data from medical records without the availability of dynamic echographic images, and consequently, there could be a detection bias. In addition, since sperm quality is an important factor for determining fertilization rate and embryonic development and the medical center where the treatments were performed is a reference for treating male-factor infertility, we chose not to assess the oocyte fertilization rate and gestational outcomes. For this reason, further research is needed on couples without associated male-factor infertility. Other limitations of this study were its retrospective design and the small number of patients. Therefore, a prospective study with a larger number of patients is warranted.
This study suggests that age alone does not reflect oocyte quality. Consequently, a simple correlation between age and the success rate of assisted reproductive technology may not be meaningful for patients with DOR up to 35 years of age. Accordingly, for better reproductive planning and to clarify optimal biological time to become pregnant, the ovarian reserve in those at risk of premature ovarian failure (even in young patients) needs to be investigated.
I would like to thank Solena Ziemer Kuzma, who helped with the statistical analysis.
No potential conflict of interest relevant to this article was reported.
This study was approval by the Institutional Review Board. The study was performed in accordance with the principles of the Declaration of Helsinki.
Written informed consent and the use of images from patients are not required for the publication.
None.
Classification of patients into groups according to normal or decreased ovarian reservea) with respect to basal follicle-stimulating hormone (FSH) levels, anti-Müllerian hormone (AMH) levels, or antral follicle count (AFC). DOR, decreased ovarian reserve; NOR, normal ovarian reserve. a)Normal basal FSH levels <10 mIU/ mL, AMH levels ≥1.2 ng/mL, and AFC ≥10 (n=49).
Oocyte quality according to maturity and morphology
Oocyte maturity |
Mature: oocytes in metaphase II |
Immature: oocytes in prophase I and metaphase I |
Oocyte morphology |
Abnormal shape |
Abnormal zona pellucida |
Abnormal perivitelline space (include changes in the polar corpuscle) |
Abnormal cytoplasm |
Characteristics of woman participants (n=49)
Variable | Mean±standard deviation | Median (minimum-maximum) |
---|---|---|
Age (yr) | 31.1±3.3 | 31 (23–35) |
Spouse age (yr) | 34.2±5.7 | 34 (24–58) |
Volume of right ovarian at US (cm3) | 6.1±2.9 | 5.4 (1.8–13.9) |
Volume of left ovarian at US (cm3) | 7.9±8.8 | 6.2 (1.1–47.7) |
AFC | 11.7±6.7 | 10 (2–27) |
Estradiol (ng/mL) | 55.3±38.4 | 44.6 (19.0–241.0) |
FSH (mIU/mL) | 7.4±2.7 | 6.5 (3.6–17.0) |
LH (mIU/mL) | 5.2±2.2 | 4.9 (1.6–11.1) |
AMH (ng/mL) | 1.9±1.6 | 1.4 (0.1–6.6) |
Height (m) | 1.7±0.0 | 1.7 (1.6–1.8) |
Weight (kg) | 64.6±8.2 | 63.7 (51.0–89.0) |
BMI (kg/m2) | 23.2±2.5 | 22.9 (18.7–29.4) |
FSH/hMG dose (IU) | 2,245±614 | 2,145 (1,025–3,850) |
Infertility time (yr) | 2.9±2.3 | 2 (1–10) |
Number of mature oocytes | 5.5±3.4 | 5 (1–14) |
Number of immature oocytes | 1.5±1.8 | 1 (0–7) |
Number of immature oocytes in prophase I | 0.8±1.3 | 0 (0–5) |
US, ultrasound; AFC, antral follicle count; FSH, follicle-stimulating hormone; LH, luteinizing hormone; AMH, anti-Müllerian hormone; BMI, body mass index; hMG, human menopausal gonadotropin.
Serum basal FSH levels when decreased ovarian reserve was defined according to AMH levels or AFC
Patients (n=49) | Basal serum FSH levels (mIU/mL) | ||
---|---|---|---|
AMH (ng/mL) | <0.001 | ||
<1.2 | 19 | 8.8±2.4 | |
≥1.2 | 30 | 6.5±2.6 | |
AFC (n) | 0.021 | ||
<10 | 23 | 8.5±3.3 | |
≥10 | 26 | 6.4±1.6 |
Values are presented as mean±standard deviation.
FSH, follicle-stimulating hormone; AMH, anti-Müllerian hormone; AFC, antral follicle count.
Non-parametric Mann-Whitney
Association between variables of patients and oocyte quality according to maturity and morphology in young women aged up to 35 years who underwent controlled ovarian stimulation (n=49)
Variable | OR (95% CI) | |
---|---|---|
Oocyte immaturity | ||
Age | 1.1 (0.9–1.3) | 0.151 |
FSH | 1.2 (1.1–1.5) | 0.040 |
AMH<1.2 | 3.3 (1.2–8.8) | 0.017 |
AFC<10 | 1.2 (0.4–3.5) | 0.704 |
FSH≥10 | 4.4 (1.2–15.5) | 0.023 |
FSH/hMG dose |
0.8 (0.5–1.2) | 0.329 |
Abnormal shape | ||
Age | 1.1 (0.9–1.3) | 0.356 |
FSH | 1.1 (0.9–1.4) | 0.281 |
AMH<1.2 | 2.3 (0.8–6.8) | 0.127 |
AFC<10 | 2.9 (1.1–7.6) | 0.035 |
FSH≥10 | 1.5 (0.3–6.6) | 0.625 |
FSH/hMG dose |
0.9 (0.6–1.4) | 0.770 |
Abnormal zona pellucida | ||
Age | 1.1 (0.7–1.6) | 0.696 |
FSH | 1.4 (0.8–2.4) | 0.282 |
AMH<1.2 | 2.4 (0.2–33.6) | 0.517 |
AFC<10 | 1.3 (0.1–16.7) | 0.830 |
FSH≥10 | 2.0 (0.1–69.2) | 0.703 |
FSH/hMG dose |
2.1 (0.7–6.3) | 0.193 |
Abnormal perivitelline space | ||
Age | 1.1 (1.0–1.3) | 0.176 |
FSH | 1.1 (0.9–1.3) | 0.340 |
AMH<1.2 | 3.0 (1.2–7.6) | 0.018 |
AFC<10 | 1.9 (0.8–4.9) | 0.160 |
FSH≥10 | 1.1 (0.3–4.3) | 0.831 |
FSH/hMG dose |
1.0 (0.7–1.4) | 0.978 |
Abnormal cytoplasm | ||
Age | 1.1 (0.9–1.4) | 0.182 |
FSH | 1.4 (1.1–1.8) | 0.006 |
AMH<1.2 | 5.3 (1.7–16.7) | 0.004 |
AFC<10 | 2.1 (0.6–7.1) | 0.224 |
FSH≥10 | 3.3 (0.6–16.7) | 0.150 |
FSH/hMG dose |
1.5 (0.9–2.4) | 0.110 |
Data presented are in mIU/mL for basal FSH levels, ng/mL for AMH levels, in years for age, and in IU for FSH/hMG dose.
FSH, follicle-stimulating hormone; AMH, anti-Müllerian hormone; hMG, human menopausal gonadotropin; AFC, antral follicle count; FSH, follicle-stimulating hormone; OR, odds ratio; CI, confidence interval.
Univariate multilevel logistic regression,
Each 500 IU.