Human oocytes play an important role in determining embryo competence and, therefore, in vitro fertilization (IVF) results [1] - [3] . Oocyte quality is not only influenced by the nuclear and mitochondrial genome but also by the microenvironment provided by the ovary and the pre-ovulatory follicle that influences transcription and translation as a consequence of cytoplasmic maturity [3] - [5] . In contrast to in vivo processes, the application of ovarian hormone stimulation protocols for IVF bypasses the complicated selection procedure that usually occurs during oocyte development and maturation of a single oocyte for ovulation and allows for the maturation of many oocytes, often with compromised quality [3] [6] . It has been speculated by that some morphological irregularities that can easily be assessed at the light microscopy level may reflect a compromised developmental ability of the oocytes and could therefore represent a useful tool for selecting competent oocytes before fertilization [1] . Oocyte morphological assessment in the laboratory is first based on the presentation of the cumulus-corona cells [1] . The cumulus-corona mass should appear as an expanded and mucified layer for mature oocytes due to the active secretion of hyaluronic acid [3] [7] . This extracellular matrix molecule interposes between the cumulus cells (CCs), separating them and conferring on the cumulus-corona mass a fluffy ‘cloud-like’ appearance [3] . However, stimulated cycles may be characterized by asynchrony between the nuclear maturation status of the oocyte and the expansion of the cumulus-corona cell mass. This has been suggested to be caused by a different sensitivity of the oocyte and the cumulus-corona mass to the stimulants [1] - [3] .

Following the removal of the cumulus-corona cells in preparation for intracytoplasmic sperm injection (ICSI) [3] [8] , oocyte evaluation is more accurate and is based on the nuclear maturation status, the morphology of the cytoplasm, and the appearance of the extracytoplasmic structures [2] [3] [6] [9] . The presence of the first polar body (PB) is normally considered to be a marker of oocyte nuclear maturity [2] . However, studies using polarized light microscopy have shown that oocytes displaying a polar body may still be immature [10] - [12] . Only those displaying a meiotic spindle (MS) can be considered as true, mature, Metaphase II (MII) stage oocytes [11] . The presence, position, and retardance of MS have been suggested to be related to the developmental competence of the oocyte [11] . In a meta-analysis, however, only in vitro development can be related to the morphology of the MS. Analysis of in vivo development is relatively rare in the literature, and most meta-analyses failed to show significant differences in implantation rates between embryos derived from oocytes displaying detectable MS and those without [3] [13] . Nuclear maturity alone is, in fact, not enough to determine the quality of an oocyte. Nuclear and cytoplasmic maturation should be completed in a coordinated manner to ensure optimal conditions for subsequent fertilization [13] .

Considering that both extreme forms of ovarian response (low and hyper-response) may be associated with diminished oocyte quality [14] - [16] , it is hypothesized that lower or higher levels of AMH might negatively affect the quality of oocytes and embryo development. In Vitro Fertilization (IVF) treatments involve the application of sophisticated ovarian stimulation protocols, continuous patient monitoring, and delicate follicle aspiration, whereby mature oocytes are harvested and fertilized.

Available evidence mostly relates to reported outcomes from patients in the developed world, including Europe and the United States. The present study aimed to assess various limiting factors, including ovarian reserve testing, to serve as a guide for controlled ovarian stimulation to obtain high-quality oocytes. AMH, despite being a good marker of ovarian response to Controlled Ovarian Stimulation (COS) that predicts oocyte quantity, has failed to predict the quality of oocytes in ART programs. Previously published reports have focused on the relation between AMH and oocyte quantity but not the nuclear maturity of oocytes [17] [18] . Hence, the importance of conducting this research.

Data analysis was performed using Spearman’s rho correlation coefficient. An initial descriptive study was conducted. The correlation between AMH, age, and the quantity of eggs retrieved was determined using Spearman’s rank correlation coefficient. Results showed a positive correlation between AMH and the number of metaphase II oocytes retrieved. Higher AMH levels were associated with more Metaphase II oocytes obtained, while lower AMH levels were associated with lower ovarian response and fewer mature (metaphase II) oocytes obtained. However, there was a strong negative correlation between AMH levels and the age of women. All p-values < 0.001 were considered statistically significant. Anti-Müllerian hormone levels and the number of oocytes obtained in the study groups and cycle outcomes according to AMH were plotted as bar charts ( Figure 1 , Figure 2 ). Subgroup analysis was performed according to five AMH ranges: group 1: ≤ 0.3 ng/mL (probably negligible response); Group 2: > 0.3 ≤ 2.19 ng/mL (expected lower response); Group 3: > 2.19 ≤ 4.00 ng/mL (possibly intermediate response); Group 4: > 4.00 and ≤ 6.79 ng/mL (normal response); and Group 5: ≥ 6.79 ng/mL (high response) were plotted as bar chart showing specific dysmorphism in the oocytes ( Figure 3 ).

Four hundred and twenty-six (426) patients were admitted to IVF/ICSI treatment from January 2017 to December 2019. The data presented were limited to the first

IVF cycles to minimize repeated-measures bias. Eight (8) cycles were cancelled due to poor ovarian response. Varying predicted human oocyte maturity grading based on morphological characteristics in terms of the cumulus-oocyte-complex was observed among patients ( Table 1 ). Summarized patients’ social demographics, cycle characteristics, and controlled ovarian stimulation (COS) outcomes according to AMH levels of the participants, age, duration of stimulation, antral follicles, total dose of gonadotropin, follicles ≥ 18 mm on hCG day, number of total oocytes retrieved, and cancelled cycles are recorded ( Table 2 , Table 3 ). Although the ages of women were found to be an independent predictor of ovarian reserve, there were no significant differences between groups concerning body mass index (BMI). Observed variables shown in Table 3 included patient age; serum AMH and antral follicle count (AFC) at the start of the cycle, number of stimulation days; number of follicles ≥ 18 mm on hCG day; number of oocytes retrieved and of metaphase II oocytes. The remaining 418 retrieval cycles were divided into five subgroups according to AMH categories: group 1 ≤ 0.30 (n = 20), group 2 > 0.30 ≤ 2.19 (n = 132), group 3 > 2.19 ≤ 4.00 (n = 158), group 4 > 4.00 ≤ 6.79 (n = 73), and group 5 ≥ 6.79 (n = 35) as indicated in ( Table 4 ). Patients were divided into five subgroups according to AMH categories about oocyte dysmorphisms: group 1(10). Group 2(12), group 3 (24), group 4 (124), and group 5 (792) are shown in Table 5 . Table 6 and Table 7 show Spearman’s rank correlation coefficient between Age, AMH, and the number of oocytes retrieved, quality of metaphase II oocytes, with p-values.

The grading takes into account the cumulus compactness, corona density, and cytoplasm granularity of the oocyte if visible [20] .

Data are presented as mean values (± standard deviation); AMH: Anti-Müllerian hormone (ng/mL); FSH: Follicle Stimulating hormone; AFC: Antral Follicle Count; BMI: Body Mass Index; hCG: Human Chorionic Gonadotropin.

SD = Standard Deviation.

**Correlation is significant at the 0.01 level (2 2-tailed).

There is a strong negative correlation between AMH and the age of women.

In this study, we assessed the association between AMH levels and oocyte quality (metaphase II oocytes retrieved). The quantity and quality of oocytes obtained after egg retrieval have been regarded as variables that influence fertilization, embryo development, and implantation of derived embryos [14] [21] . The relation between AMH levels and the number of metaphase II oocytes obtained in each group was recorded in Table 4 . A significant correlation between AMH levels and ovarian response was observed in the number of oocytes retrieved during COS. Our results show that age and AMH levels are the superior parameters predicting ovarian response. However, even in women of comparable age and AMH levels, there was a wide variation in the individual ovarian reserve [22] - [25] . AMH is a very promising and possibly the best actual candidate to evaluate individual ovarian response to gonadotropin stimulation and to detect poor responders with levels of AMH ≤ 0.30 ng/mL.

Considering that both extreme forms of ovarian response (low and hyper-response) may be associated with diminished oocyte quality [14] - [16] , it is hypothesized that lower or higher levels of AMH might harm the number of oocytes and embryo development. Subgroup analysis of the five AMH categories showed that group 1 (22) patients, 6 cycle cancellations (27.27%), 130 MII oocytes, group 2 (138 patients), 2 cycle cancellations (1.45%), 298 MII oocytes, group 3 (158 patients), no cycle cancellations, 890 MII oocytes, group 4 (73 patients), no cycle cancellation, 1734 MII oocytes and group 5 (35 patients), 1224 MII oocytes with no cycle cancellations. AMH levels ≤ 0.30 ng/mL (patients with poor prognosis) were observed in 16 patients (3.83%) in group 1. Patients within this AMH level range accounted for 27.27% of cancelled or failed cycles among the study groups.

Though the total number of oocytes retrieved in group 5 was higher (2016) than those in group 4 (1858), the total number of mature (metaphase II) oocytes obtained in group 5 was less (1224) as compared to those in group 4 (1734). This could be attributed to a hyper-response to COS in group 5 that is associated with diminished oocyte quality [26] [27] . Initial data obtained in chapter 2 of this study confirmed the reduced number of metaphase II oocytes obtained in group 5: women with AMH values greater than median value of 3.5 ng/mL, mean value of 4.85 ng/mL, a standard deviation of 3.34 ng/mL and AMH levels ≥ 6.75 ng/mL (group 5). These patients were considered “high responders” with PCOS-like features without meeting the full diagnostic criteria for exclusion. In assisted reproduction, women with high AMH, especially those with PCOS, are more likely to be “high responders” to gonadotropin stimulation. While this can increase egg yield, it also raises the risk of ovarian hyperstimulation syndrome (OHSS), a potentially serious complication [28] [29] . OHSS is a notable iatrogenic complication in IVF treatment with the retrieval of usually more than fifteen (15) oocytes with a high number of immature oocytes and smaller numbers of mature (metaphase II oocytes) following a standard COS protocol [15] . IVF patients with PCOS constitute 20% of clients undergoing COS, and less than one-fifth of them present symptoms of OHSS [30] . Increased levels of serum AMH in PCOS patients have been suggested to be a result of excess induction of androgens in small antral follicles [30] - [32] . It has been previously described that when cumulus cells are denuded for ICSI, more than 60% of all oocytes show at least one abnormal morphological feature [33] - [35] . It is unclear whether the correlation between AMH and the presence of oocyte abnormalities observed was due to decreased secretion by granulosa cells in poor-quality oocytes [36] or as a result of a possible detrimental effect of low levels of AMH on oocyte quantity and quality [14] [17] [21] . The list of dysmorphic markers associated with reduced quality and development of oocytes was observed and recorded. Oocyte dysmorphisms such as refractile bodies in ooplasm may affect fertilization and embryo quality [37] , while the presence of vacuoles in ooplasm, according to the literature, has been found to reduce fertilization rate in an ICSI study [37] . Whether or not disturbances occur may mainly depend on the size of the vacuole; it is yet to be established. The presence of smooth endoplasmic reticulum (sER) clusters may be indicative of a disturbance that may affect embryo development since the clinical pregnancy rate was much lower in oocytes with sER clusters compared to those without sER clusters [6] . In addition, a baby born from a cluster-positive oocyte that was fertilized had Beckwith-Wiedemann syndrome, a disease associated with disturbed expression from an imprinted gene [6] [38] is a long debate on the predictive value of the size and morphology of the first polar body (PB).

A study by Ebner et al. reported that oocytes with normal-sized, intact first-polar bodies produced embryos with a higher success rate of conception cycles. Oocytes with fragmented PBs had a significantly lower blastocyst formation rate [6] [36] [38] . The fragmentation rate is likely to increase over time after egg retrieval; this may explain the negative results found in two other studies where the timing of ICSI and PB morphology analysis differed [39] . There was no link between PB shape and the chromosomal makeup of PBs in oocytes from older patients [11] [40] . Mouse models suggest that very large PBs indicate loss of cell cycle control and disrupted spindle function [40] - [42] . Further research is needed to determine if this also applies to humans. The rapid degeneration of the first PB and potential differences in maturation kinetics influenced by maternal age and stimulation protocols make it challenging to evaluate the usefulness of PB scoring for oocyte selection.

Clear, colorless cytoplasm is considered a marker of good quality in oocytes, while extensive cytoplasmic granularity, particularly central granulation, was negatively correlated with a conception cycle and was associated with increased aneuploidy in embryos [43] . The presence of dark cytoplasm in oocytes decreased by 83% the likelihood of obtaining a good quality embryo and thus may be considered negatively correlated with oocyte quality [43] - [45] . Increased cytoplasmic viscosity is also considered a marker of low oocyte quality but is difficult to quantify objectively [3] [46] [47] . Funnel persistence as a result of high viscosity can only be analyzed in ICSI cycles after the microinjection of sperm and can thus not be used for oocyte selection before fertilization. Oocytes with larger than larger-than-normal perivitelline space were associated 1.8 times higher chance of having good-quality embryos compared to those with perivitelline dysmorphisms [2] . Perivitelline granularity was not predictive of fertilization and cleavage rate, but rather a physiological maturation-dependent phenomenon related to stimulation protocol [3] [38] [47] . Zona pellucida (ZP) is one of the cellular constituents of the oocyte that is synthesized during oocyte growth and maturation. The relative order of fibers might reflect a continuously favorable environment during a period of oocyte development when it acquires nuclear and cytoplasmic maturational competence. Several studies have focused on measuring zona thickness, but differences between groups were always fairly low and therefore not very useful for selecting oocytes [48] - [50] . The study showed a mean (SD) (14.15) of zona pellucida abnormalities in the oocytes obtained. In a retrospective analysis of the mean retardance of light by the three layers of the zona pellucida in a line scan across the zona from oocytes transferred after ICSI showed that the mean magnitude of light retardance was nearly 30% higher (p < 0.001) in the inner layer of the zona pellucida of oocytes contributing to a conception cycle compared to a non-conception cycle [49] . The magnitude of light retardance by the zona pellucida inner layer appears, therefore, to present a unique non-invasive marker for oocyte developmental potential and/or cycles with high probability of success. A study employing the same techniques fully confirmed the finding by showing a correlation between the mean retardance of the human oocyte spindle and the zona developmental potential of the oocytes [40] [42] . Indeed, AMH might play an important role in primordial follicle selection and cyclic growing follicle recruitment [51] . Moreover, AMH might regulate the selection of the dominant follicle through the inhibitory effects of AMH on the initial recruitment of primary follicles from the resting primordial follicle pool and through the regulation of FSH sensitivity in the human ovary [52] .

In summary, the results confirm the relevance of AMH measurement as an important screening test for reduced ovarian reserve in women. Furthermore, a level of AMH ≤ 0.3 ng/mL correctly predicts a very poor response with two oocytes in some cases and a poor outcome even in groups of high age-related heterogeneity. If AMH levels are ≤ 0.30 ng/mL, an antral follicle count should be added to exclude false positive results and increase specificity, especially in a routine clinical setting with heterogeneous groups of patients. Chronological age fairly well predicts ovarian response and the upcoming perimenopausal and menopausal transition. Important studies have shown that AMH is a predictor for the occurrence of perimenopausal transition (cycle length irregularity) within 3 - 5 years [15] at levels of 0.92 ng/mL.

Our study affirms that by early detection of women with a reduced ovarian response, using a cut-off level < 0.30 ng/mL, treatment options are still available (high-dose stimulation in IVF, poly-ovulation) with favorable pregnancy rates, not statistically different from those of women with ‘normal’ ovarian reserve. Therefore, women who want to get pregnant should be advised to get AMH levels determined if they do not achieve pregnancy after six cycles of regular intercourse. AMH is a suitable screening test and may replace FSH, which gains relevance only in the late reproductive phase. AMH plays a major role in assisted.

Reproductive technology allows not only the quantification of the ovarian reserve but also the prediction of eventual ovarian response to IVF stimulation.

Although AMH is good for predicting oocyte quantity, the age of women remains the strongest predictor of oocyte quality. To evaluate or complement the prediction of oocyte quality, different or complementary markers, both biochemical and clinical, such as AFC, day 3 FSH, and Estradiol, mitochondrial biomarkers, serum progesterone in the late follicular phase, and Preimplantation Genetic Diagnosis Testing for Aneuploidy (PGT-A) have shown promise to better predict oocyte quality. Cumulus cell gene expression and other markers like HAS2, PTGS2, and GREM1 have also shown promise, mainly in research but not in routine or clinical use yet.

The association between AMH and the quality of human oocytes observed in our fertility center in this study should be interpreted with caution due to several limitations. The single-center design restricts the diversity of clinical practices, laboratory protocols, and patient demographics, reducing the generalizability of the findings. Results may not apply to other fertility centers within Ghana or internationally, as they are influenced by the specific genetic, environmental, lifestyle, and socioeconomic characteristics of the studied population. Additionally, cultural and healthcare access differences may further limit extrapolation. Larger, multicenter studies across varied populations are needed to confirm and extend these observations to broader contexts.

Our study indicates that anti-müllerian hormone is a dependable measure for predicting oocyte quality in Ghanaian women undergoing IVF treatment, but it is not a reliable indicator of oocyte nuclear maturation. Our findings show that low AMH levels alone are not appropriate for withholding fertility treatment, and further research is necessary to understand the link between AMH and oocyte nuclear maturation.

D.M., M.M.M. conceived and designed the study; M.M.M. collected the data with supervision from D.M. M.M.M. and D.M. conducted the data analysis; D.M. and G.K.N. Authored the article with significant contributions from M.M.M. All authors approved the final manuscript. All authors have read and agreed to the published version of the manuscript.

Ethical approval for this study (number: CHRPE/AP/276/17) was provided by the Committee on Human Research, Publication, and Ethics (CHRPE) of the School of Medicine and Dentistry, College of Health Sciences, Kwame Nkrumah University of Science and Technology (KNUST). We obtained written consent from all participants before the data collection.

Not applicable. Data Availability Statement: The dataset for this study will be made available upon reasonable request from the corresponding author.

We are extremely grateful to all the staff of Airport Women’s Hospital for their support and cooperation during the recruitment and data collection. We are also grateful to all the women who consented to participate in this study.