Evaluation of assisted reproductive technology treatment outcomes based on stimulation dosages and anti-Mullerian hormone levels

Introduction

Infertility is a clinical condition, in which there is a failure to achieve pregnancy even after unprotected intercourse for a period of 12 months. Globally, the prevalence of infertility is estimated at 8–12% for women aged 20–44 years old, with 1 in 6 couples experiencing some form of infertility problems (ART fact sheet, European Society of Human Reproduction and Embryology). Recently, in Malaysia the total fertility rate of women of reproductive age in Malaysia has declined to 1.7 babies in 2020 as compared to 1.8 in 2019.¹ One of the hallmark causes of infertility in the majority of the population is delaying conception to the late thirties or in early forties.² Physiological ovarian aging is referred to as an age-mediated decrease in functional ovarian reserve within expected ranges. Previous reports have shown that female ovarian reserves decrease with substantial increases in age. The ability of fertilization during natural and stimulated ovarian cycles declines with maternal age, and thus optimal fertility is achieved in the age range between 20–30 years old.³ Globally, recent socio-economic changes have led to increases in infertility rates and there have also been increased numbers of women approaching for assisted reproductive technology (ART) treatments. While mounting treatment strategies are employed in the management of infertility, such as intrauterine insemination (IUI), to in vitro fertilization (IVF), intra-cytoplasmatic sperm injection (ICSI), testicular sperm extraction (TESE) and preimplantation genetic testing (PGT), ART still remains the gold standard for controlled ovarian stimulation.⁴

Various markers of ovarian reserve, which determines the quantity of primordial follicles and oocyte quality, have been established as predictors of ovarian response.⁵ Among the biochemical parameters, such as basal follicle stimulating hormone (FSH), estradiol and inhibin levels may differ during the menstrual cycle and thus their diagnostic utility is limited.⁶ Imaging modality using transvaginal ultrasonography is used to evaluate the antral follicle count (AFC) but its sensitivity is affected by sonographers' intra- and inter-observer reproducibility.⁷

Anti-Müllerian hormone (AMH), a dimeric glycoprotein that belongs to the transforming growth factor-β (TGF-β) family, has been determined as an effective surrogate marker in the evaluation of functional ovarian follicle reserve.⁸ AMH is synthesized by granulosa cells of the preantral and small antral follicles and also serves as a follicular gatekeeper by blocking the initial follicle recruitment and FSH-mediated growth and selection.⁹ When compared with other ovarian reserve biomarkers, AMH displays high sensitivity for stability and good measurement repeatability. Earlier reports have shown the diagnostic accuracy of AMH levels in the prediction of high and poor responses during gonadotropin-releasing hormone (GnRH) agonist or GnRH antagonist protocols^10,11 and also in AMH-mediated ovarian stimulation.¹¹ Safe and effective ovarian stimulation is a pivotal step in the success of ART treatment cycles. It is important to categorize patients who plan to undertake ART as normal, poor or high responders, thus choosing the appropriate dosage of gonadotropin for every patient that could yield a suitable number of oocytes.¹² In this backdrop, the present study was performed to evaluate the FSH and Human Menopausal Gonadotropin (HMG) dosages on the outcomes of ART in low, normal and high responders.

Methods

Results

This prospective study was conducted on 191 patients who met the inclusion criteria. The basic demographics and clinical characteristic of the participants are shown in Table 1.²⁷

Regarding age distribution, the majority of the participants were aged between 30–35 years old (40.8%), followed by 36–40 years old (34%). The results are shown in Table 2.

AMH distribution among the study participants is shown in Table 3. According to the AMH levels, the majority of the patients were normal responders (AMH:5.5–24.9 pmol/L), which constituted 52.4% of patients, high responders (AMH: ≥ 25 pmol/L) encompassing 29.8% of patients and low responders (AMH ≤ 5.4 pmol/L), which constituted 17.8% of patients.

Association between AMH levels and fertility parameters

A positive correlation (r=0.617) was found between AMH and number of oocytes retrieved, which was statistically significant (p=0.01). Likewise, a positive correlation (r=0.545) was found between AMH and number of oocytes sucessfully fertilized and the association was statistically significant (p=0.01). However, the corelation found between AMH levels and number of embryos utilized was not statistically significant (r=0.08; p=0.23). The results are shown in Figure 1 and Figure 2.

Figure 1. Association between AMH levels (pmol/L) and number of oocytes retrieved.

AMH, anti-Müllerian hormone.

Figure 2. Association between AMH levels and number of oocytes successfully fertilized.

AMH, anti-Müllerian hormone.

Association between age and AMH levels are shown in Figure 3. A negative correlation (r=-0.391) exists between age and AMH levels (as age increases AMH levels decrease) and the association was found to be significant (p=0.01).

Figure 3. Association between AMH levels and age.

AMH, anti-Müllerian hormone.

Discussion

The present study confirmed the usefulness of AMH as a reliable biomarker of ovarian function. A mounting number of studies have shown that AMH is a new technique for the evaluation of ovarian reserve. AMH levels decrease throughout a woman’s reproductive life.¹⁴ Serum AMH levels on day three of the menstrual cycle display marked reduction with age and show marked association with AFC. During spontaneous menopause, undetectable AMH concentration have been observed.¹⁵ In women with normal menstrual phase, oophorectomy leads to the disappearance of AMH in three to five days, and thus substantiates that circulating AMH is attributed to ovarian origin.¹⁶ AMH is a reliable endocrine marker, which provides information about the transition of resting primordial follicles to growing follicles. AMH levels show a decreasing pattern five years before the last menstrual cycle and thus showcases the biological event of menopause.¹⁷

In the present study, based on the AMH levels the patients were categorized as low, normal and high responders. We observed a high prevalence of normal responders, with 52.4% as compared to high responders (29.8%) and low responders (17.8%). In the current study, the average FSH per day was significantly higher in low responders as compared to high and normal responders. Earlier trials showed that the oocyte yield can be upregulated using higher doses of FSH,^18,19 thus in routine clinical practice, the low responders must be treated with higher doses as compared to the standard FSH dose of 150 IU/day (from 225 to 600 IU/day) to increase the number of retrieved oocytes. Similarly, the average HMG per day was significantly higher in low responders as compared to high and normal responders. There has been no marked difference between HMG and FSH for ovarian stimulation with respect to intermediate outcome and ART surrogate markers. Further for both HMG and FSH, the outcomes are similar for Metaphase II (MII) matured oocytes, frequency of high-quality embryos, zona pellucida morphology, and polar body evaluation.²⁰ Previous reports have shown that there are no marked effects in the number of oocytes retrieved with HMG, however mounting studies indicate that there has been minimal decrease in the number of oocytes retrieved (typically around one oocyte less per retrieval).²¹

In the present study, oocytes retrieved, retrieval rate and blastulation rate were significantly higher in high responders compared with low responders. To some extent, the results shown were anticipated as AMH has been used an indicator of oocyte reserve in previous studies, whereas the resulting fertilized or transferred embryos may be due to a chance process based on many various factors such as quality of oocyte and sperm.²² Furthermore, in our study there were no significant differences in the maturation rates, fertilization rates and embryo utilization rates among the low, normal and high responders. Similarly in a study conducted by Umarsingh et al.,²³ there was no statistically significant relationship between numbers of oocytes fertilized vs. AMH category (p>0.05).

In the present study, there was a positive significant relationship between AMH and number of oocytes retrived and number of oocytes sucessfully fertilized. Similar to the present study, Kotanidis et al.,²⁴ reported that there was a strong, positive correlation between AMH and the number of collected oocytes. Furthermore, Wiweko et al.,²⁵ also reported that serum AMH levelsarethe signficant predictor of oocyte quality. Meanwhile, we have observed non-significant associationsbetween AMH levels and the number of embryos utilized. Our study corroborates with the findings of Umarsingh et al.,²³ where there was no signficant relationship between AMH and embryo utilization.

The Pearson’s correlation test found an R-value of -0.328 between AMH and age, thus showing that there was a negative association, which was found to be significant (p=0.01). A stronger relationship between these two variables was expected as it is known that as age increases, AMH should decrease. Also, itis suggested that serum AMH is identified as the improved endocrine marker to measure the reproductive capability in advanced age. Similarly, Ishii et al.,²⁶ reported an inverse moderate correlation (correlation coefficient r=-0.5, p<0.01) between age and AMH levels.

Conclusions

In conclusion, the study outcome revealed that AMH serves as a reliable marker to evaluate ovarian reserve and may benefit women with advanced age planning for pregnancy. Albeit AMH hormone serves as an effective endocrine marker however, its valuable role in the assessment of decline in the ovarian pool must be compared with other markers. AMH can also significantly predict the number of oocytes retrieved and number of oocytes sucessfully fertilized. Further, the study findings also confirmed that age displayed signficant assocaition with AMH levels. In addition, the rates of retrieval, maturation, fertilization, embryo utilization and blast were higher among the high responders as compared to low responders. Thus, increase in dosages of ovarian stimulation protocol is required among the low responders to achieve quality embryos. During counseling, the level of AMH is important to educate patients regarding the high dose of medication administration in order to achieve a good outcome.

Data availability