Investigating the correlation between AMH and the number of high-quality embryos from IVF/ICSI in 30-44 year-old infertile women

doi:10.21203/rs.3.rs-1250366/v1

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Investigating the correlation between AMH and the number of high-quality embryos from IVF/ICSI in 30-44 year-old infertile women

https://doi.org/10.21203/rs.3.rs-1250366/v1

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Background To explore the effect of anti-Müllerian hormone (AMH) on embryonic development by studying the relationship between serum AMH concentration and the high-quality embryo number during the first cycle of in vitro fertilization (IVF) /intracytoplasmic sperm injection (ICSI) in 30-44 year-old infertile women.

Methods A retrospective cohort study of 335 cycles was conducted. The study population was the patients who received the first IVF/ICSI cycle at the Reproductive Medicine Center of * * * * * * from January 2019 to December 2020. The basic characteristics, clinical medication and cycle outcome of patients were collected, and the correlation between AMH and the number of high-quality embryos was analyzed.

Results After adjusted for confounding factors (female age, AFC and COH protocol), sensitivity analysis showed that high-quality embryo number in high AMH group increases by 2.2 units for each additional unit of AMH (≥8.5 ng/mL) (95%CI: (0.8, 3.7), P=0.003). The two-piecewise regression model identified the inflection point as AMH=7.1 ng/mL, and the effect size, 95% CI and P value on both sides of the inflection point were 0.5(0.3, 0.7), <0.001 and 0.1(-0.1, 0.2), 0.518, respectively. The results of stratified analysis and interaction test showed that the relationship between AMH and the high-quality embryo number was stable among different subgroups (P>0.05).

Conclusion When AMH<7.1ng/mL, there is a positive correlation between AMH and the number of high-quality embryos in this study population. Therefore, the effect of age-related ovarian reserve biomarker AMH on reproductive prognosis can provide reference for ART clinical treatment.

Plain English summary Diminished ovarian reserve (DOR) refers to a decrease in the number and/or quality of oocytes in the ovary, accompanied by a decrease in anti-Müllerian hormone (AMH) levels, a decrease in antral follicle count (AFC) and an increase in follicle stimulating hormone (FSH) levels. The most common clinical symptoms are shortened menstrual cycle and decreased menstrual flow. Most patients go to the hospital after long-term unsuccessful attempts to conceive, so delays in diagnosis are common. Assisted reproductive technology (ART) is the main means of achieving pregnancy in patients with DOR. With the improvement and adjustment of the ovulation induction program and the innovation of laboratory technology, the clinical pregnancy rate of ART has increased. However, DOR patients have poor ovarian response to gonadotropins (Gn), manifested as less follicle development, low blood estrogen levels, high Gn dosage, lower-than-expected oocyte retrieval, high cycle cancellation rate, and low clinical pregnancy rate in ovarian stimulation cycles. Therefore, this type of patients is a difficult point in the field of assisted reproduction. In the present study, elevated AMH was associated with an increased number of high-quality embryos in individuals aged 30-44 years at high risk for DOR. AMH can assess the potential reproductive prognosis of such patients and provide valuable data for clinical treatment of such patients.

anti-Müllerian hormone

In vitro fertilization/intracytoplasmic sperm injection

ovarian reserve

number of high-quality embryos

A key issue affecting IVF/intracytoplasmic sperm injection (ICSI) treatment is "ovarian reserve" [1]. The term "ovarian reserve" is often used to describe a woman's reproductive potential--specifically referring to the number and quality of oocytes [2]. The number of oocytes largely depends on the female age [3]. Once the female reaches 30 years old, the number of oocytes drops sharply, accompanied by significantly increased abortion rate [4]. Therefore, women between 30 to 44 years old have a higher risk of ovarian aging [5]. "Ovarian reserve" assessment is particularly important for the IVF/ICSI outcome of infertile women at this age. Clinicians can use this assessment to select controlled ovarian hyperstimulation (COH) protocol and predict the ovarian response of patient receiving assisted reproductive technology (ART) [6].

Among the many biomarkers of ovarian reserve, AMH is widely used because of its high specificity and intra-cycle and inter-cycle stability [7]. AMH is a product of preantral follicles and granulosa cells of small antral follicles. Its level begins to increase during female adolescence, reaches the peak around the age of 25, and then gradually decreases until undetectable after menopause [8]. Studies have shown that even if the women have extremely low AMH levels (<0.16 ng/mL), they still have the possibility of live birth after IVF/ICSI. Therefore, clinically, infertile patients cannot be refused for ART treatment solely based on AMH testing [9]. Two meta-analysis showed that AMH was a predictor for poor pregnancy and live birth after ART. However, in pregnant women with a history of pregnancy loss, the AMH value is not related with the ability of natural conception [10]. Therefore, AMH is controversial in predicting the pregnancy outcome after IVF/ICSI in infertile women [11–13], indicating that AMH is more likely a quantitative rather than a qualitative marker of oocytes.

The studies from Lv Hong and Chen Shiping both showed that embryo quality and quantity are the main predictors for successful implantation and live birth [14, 15]. Therefore, embryo quality is also critical for the outcome of ART [16, 17]. So far, there are few studies exploring the predictive value of AMH for embryo quality and the prognosis of reproduction. This study analyzed the relationship between AMH and the number of high-quality embryos in the first IVF/ICSI cycle of 30-44 year-old infertile women, explored the influence of AMH on embryonic development, and provided valuable data for the clinical treatment of such infertile patients.

1. Study population and data collection

This is a retrospective cohort study containing 335 cycles. Patient information comes from infertile women who underwent IVF/ICSI treatment between January 2019 and December 2020 at the Reproductive Medicine Center of * * * * *. Figure 1 is the flowchart of patient selection. A total of 304 patients were included into this study. The patients were 30-44 years old. The COH protocol of the first ART cycle was GnRH-A, Mild/PPOS and Ultra-Short GnRH agonist. Patients with the following conditions were excluded from the study: no oocyte was retrieved in this cycle or donor oocytes/sperm was used; patient suffered from polycystic ovary syndrome, hyperprolactinemia, thyroid disease, metabolic abnormalities or endocrine diseases such as diabetes and Cushing’s syndrome; patient with a history of ovarian surgery; patient with infectious diseases, autoimmune diseases, heart disease, etc. All data were anonymous, so the informed consent was not required.

Data collection: (1) Demographic variables: female age and male age; (2) Clinical and medical variables: female body mass index (BMI), duration of infertility, type of infertility (primary infertility/ secondary infertility), basal follicle stimulating hormone (bFSH), basal luteinizing hormone (bLH), basal estradiol (bE₂), antral follicle count (AFC), anti-mullerian hormone (AMH), male sperm concentration, sperm progressive motility, and progressive motile sperm count. (3) Clinical medication: total days of stimulation (days), total gonadotrophin (Gn) dose, COH protocol, number of follicles ≥16 mm on trigger day, number of follicles ≥14 mm on trigger day, and E₂ level on trigger day; (4) Cycle outcome: number of retrieved oocytes, number of mature oocytes (MII), number of normally fertilized oocytes in IVF/ICSI, number of high-quality embryos (including cleavage stage embryos and blastocysts).

2. COH protocol [18]

GnRH antagonist (GnRH-A) protocol: patients were given r-hFSH or urofollitropin for injection (Lishenbao) at 150-300IU/d (according to the patient's ovarian reserve and BMI) on the second or third day of the menstrual cycle; after 5 days, the Gn dose was adjusted according to the results of follicle monitoring and serum hormone test. When the dominant follicle reached about 14mm, the GnRH antagonist (cetrorelix) was given at 0.125-0.25mg/d until the human chorionic gonadotropin (hCG) trigger day.

Micro stimulation (Mild) or ovulation stimulation under high progesterone state during the follicular phase (PPOS) protocol: patients were given clomiphene (CC) 50mg/d or medroxyprogesterone 8mg/d and Gn (Lishenbao/HMG) 150-225IU on the 3rd day of the menstrual cycle until the day of hCG injection. Follicle monitoring and serum hormone level detection were performed 5 days later, and Gn dose was adjusted according to ovarian response. When there were 3 follicles with a diameter of ≥16 mm, or 2 follicles with a diameter of ≥17 mm, or 1 follicle with a diameter of ≥18 mm, patients were given Azer 250IU or hCG 10000 U for trigger.

Ultra-Short GnRH agonist protocol: GnRH agonist 0.05mg/d was given to patients on the second day of the menstrual cycle, and rFSH150-300IU was given at the same time since the third day until the day of hCG injection. When there were 3 follicles with a diameter of ≥16 mm, or 2 follicles with a diameter of ≥17 mm, or 1 follicle with a diameter of ≥18 mm, patients were given Azer 250IU or hCG 10000 U for trigger.

Oocyte retrieval was performed 36-37h after the trigger, and IVF/ICSI was performed according to clinical instructions [19]. The development of embryos was monitored every day. High-quality cleavage stage embryos and high-quality blastocysts were vitrified and cryopreserved [20].

3. Embryo morphology assessment

At 16-18 hours after IVF/ICSI, the oocyte fertilization was evaluated, and double pronucleus indicated normal fertilization. The embryonic development was evaluated according to the cleavage speed, synchronization, number of blastomeres, symmetry, spatial arrangement, and debris amount. On the third day after fertilization, the embryos with 7-10 uniform-sized blastomeres and no cytoplasmic fragments were rated as Grade I embryos; the embryos with 7-10/6/>10 blastomeres and cytoplasmic fragments <10% were rated as Grade II embryos. Grade I and II embryos were high-quality cleavage stage embryos [21]. The embryos at 5-6 days after fertilization were blastocysts. According to Gardner et al, the blastocysts with many tightly bound inner cell clusters and a large number of trophoblasts that formed a tightly arranged epithelial cell layer (≥3BB) were rated as high-quality blastocysts [22].

4. Serological analysis

Blood samples were collected from the patients on the 3rd day of the menstrual cycle and on the trigger day. The serum concentrations of bFSH, bLH, bE₂ and E₂ on the trigger day were detected by chemiluminescence measurement (Abbott Biologicals B.V., The Netherlands). The lower limits of sensitivity were: bFSH=0.06 mIU/mL, bLH=0.09 mIU/mL, E2=10 pg/mL. The serum concentration of AMH was measured by automatic chemiluminescence detection analyzer (Shenzhen Yahuilong Biotechnology Company, iFlash 3000) and AMH detection kit (acridin ester direct chemiluminescence method) (Shenzhen Yahuilong Biotechnology Company). The lower limit of sensitivity was 0.01ng/mL, and the inter-lot coefficient of variation was less than 15%.

5. Statistical Analysis

We performed statistical analysis using AMH as the target independent variable and high-quality embryo number as the dependent variable. Continuous variables were expressed as mean ± standard deviation (normal distribution) or median (quartile) (non-normal distribution), and categorical variables were expressed as frequency or percentage. Differences between groups were compared using one-way analysis of variance (ANOVA) and chi-square test. The variables in the skewed distribution were tested using the Kruskal-Wallis method. Univariate linear regression model was used to evaluate the association between AMH and the number of high-quality embryos. Three models were constructed, and multiple regression equations were used to verify the relationship between AMH and high-quality embryo number from different angles. Generalized Additive Model (GAM) was used to identify the nonlinear relationship between AMH and the number of high-quality embryos. A piecewise regression model was used to calculate the inflection point of the threshold effect of AMH on high-quality embryo number. A stratified linear regression model was used for subgroup analysis. The modification and interaction between subgroups were tested by likelihood ratio. Statistical software packages R (http://www.R-project.org, the R Foundation) and Empower (R) (www.empowerstats.com, X and Y Solutions, Inc. Boston, MA, USA) were used for data analysis. Two-sided P<0.05 indicates statistical significance.

Patient baseline characteristics

A total of 304 IVF/ICSI cycles were included in this study. According to the two clinical cut-off points of AMH, patients were divided into three groups: low AMH group (<0.7 ng/mL, n=29), medium AMH group (>=0.7, <8.5 ng/mL, n=256) and high AMH group (≥8.5 ng/mL, n=20). There was no statistical difference between the three groups in male age, duration of infertility, BMI, bE₂, male sperm concentration, sperm progressive motility, and the progressive motile sperm count. Compared with the the low AMH group, the higher AMH was related to the decrease in female age (P=0.030), type of infertility (P=0.032), decrease in bFSH (P<0.001), increase in bLH (P<0.001), increase in AFC (P<0.001), increase in total days of stimulation (P<0.001), increase in total gonadotrophin dose (P<0.001) and COH protocol (P<0.001). With the decrease of AMH, the number of retrieved oocytes (P<0.001), the number of MII oocytes (P<0.001) and the number of high-quality embryos (P<0.001) decreased significantly (Table 1).

Table 1

Baseline characteristics of the patients
AMH (ng/mL)	<0.7	>=0.7, <8.5	>=8.5	P-value	P-value*
N	29	256	20
Female age (years)	34.0 ± 4.2	34.0 ± 3.8	31.7 ± 2.3	0.030	0.023
Male age (years)	35.3 ± 7.1	34.0 ± 4.7	32.5 ± 3.1	0.149	0.534
Duration of infertility (years)	3.17 (2.33-5.33)	3.00 (2.00-6.00)	3.75 (2.75-4.62)	0.733	0.548
Type of infertility				0.032	-
Primary infertility	18 (62.07%)	110 (42.97%)	13 (65.00%)
Secondary infertility	11 (37.93%)	146 (57.03%)	7 (35.00%)
BMI (kg/m²)	22.75 ± 3.64	23.09 ± 3.30	23.39 ± 3.10	0.795	0.649
bFSH (mIU/mL)	12.75 ± 6.12	8.32 ± 2.60	6.97 ± 1.76	<0.001	<0.001
bLH (mIU/mL)	3.89 (2.59-5.23)	4.03 (3.10-5.49)	5.88 (4.67-8.70)	<0.001	<0.001
bE₂ (pg/mL)	53.95 ± 23.18	49.32 ± 19.41	43.69 ± 21.71	0.208	0.165
AFC (n)	3.00 (2.00-6.00)	11.50 (7.00-17.00)	34.50 (26.25-41.00)	<0.001	<0.001
Sperm concentration (million/mL)	61.49 (25.28-84.54)	48.42 (28.86-82.72)	39.61 (32.58-90.44)	0.380	0.760
Sperm progressive motility (a+b) (%)	0.45 ± 0.22	0.40 ± 0.18	0.39 ± 0.13	0.399	0.516
Progressive motile sperm count (a+b) (million)	65.84 (15.51-136.86)	58.64 (27.82-98.31)	48.38 (21.47-94.16)	0.287	0.774
Total days of stimulation (days)	6.00 ± 3.35	7.41 ± 1.88	8.10 ± 1.74	<0.001	0.027
Total gonadotrophin dose (IU)	1125.00 (750.00-1575.00)	1650.00 (1350.00-1950.00)	1725.00 (1481.25-2034.38)	<0.001	<0.001
COH protocols				<0.001	-
GnRH-A	3 (10.34%)	126 (49.22%)	18 (90.00%)
Mild/PPOS	19 (65.52%)	78 (30.47%)	1 (5.00%)
Ultra-Short GnRH agonist	7 (24.14%)	52 (20.31%)	1 (5.00%)
Retrieved oocytes (n)	2.00 (1.00-2.00)	5.00 (3.00-8.00)	12.00 (8.00-15.25)	<0.001	<0.001
Retrieved MII oocytes (n)	2.00 (1.00-2.00)	5.00 (3.00-7.00)	12.00 (8.00-14.25)	<0.001	<0.001
High-quality embryos (n)	1.00 (0.00-1.00)	2.00 (1.00-4.00)	6.00 (4.00-7.25)	<0.001	<0.001

Univariate Analysis

The results of univariate analysis are shown in Table 2: The female age, male age, duration of infertility, bFSH and COH protocol were negatively correlated with the number of high-quality embryos; bLH, AMH, AFC, total days of stimulation, total gonadotrophin dose, E₂ on trigger day, number of follicles ≥16 mm on trigger day, number of follicles ≥14 mm on trigger day, number of retrieved oocytes, number of MII oocytes, and the number of normally fertilized oocytes in IVF/ICSI were positively correlated with the number of high-quality embryos; the other parameters had no correlation with the number of high-quality embryos.

Table 2

Results of univariate analysis
	Statistics	effect size (β)	P value
Female age (years)	33.8 ± 3.8	-0.2 (-0.3, -0.1)	<0.001
Male age (years)	34.0 ± 4.9	-0.1 (-0.2, -0.1)	<0.001
Duration of infertility (years)	4.22 ± 3.15	-0.11 (-0.20, -0.02)	0.017
Type of infertility
Primary infertility	141 (46.23%)	ref
Secondary infertility	164 (53.77%)	0.14 (-0.44, 0.72)	0.635
BMI (kg/m²)	23.08 ± 3.31	-0.05 (-0.14, 0.04)	0.274
bFSH (mIU/mL)	8.65 ± 3.35	-0.20 (-0.28, -0.11)	<0.001
bLH (mIU/mL)	4.60 ± 2.30	0.21 (0.08, 0.33)	0.001
bE₂ (pg/mL)	49.39 ± 19.98	-0.01 (-0.02, 0.01)	0.279
AMH (ng/mL)	3.28 ± 3.07	0.42 (0.34, 0.50)	<0.001
AFC (n)	14.05 ± 11.17	0.10 (0.08, 0.12)	<0.001
Sperm concentration (million/mL)	68.08 ± 57.26	0.00 (-0.00, 0.01)	0.218
Sperm progressive motility (a+b) (%)	0.40 ± 0.18	0.82 (-0.80, 2.45)	0.323
Progressive motile sperm count (a+b) (million)	89.91 ± 116.93	0.00 (-0.00, 0.00)	0.340
Total days of stimulation (days)	7.32 ± 2.10	0.23 (0.10, 0.37)	<0.001
Total gonadotrophin dose (IU)	1629.76 ± 613.89	0.00 (0.00, 0.00)	<0.001
COH protocols
GnRH-A	147 (48.20%)	ref
Mild/PPOS	98 (32.13%)	-2.11 (-2.71, -1.51)	<0.001
Ultra-Short GnRH agonist	60 (19.67%)	-2.18 (-2.88, -1.47)	<0.001
E₂ day of trigger (pg/mL)	2126.19 ± 1933.21	0.00 (0.00, 0.00)	<0.001
Number of follicles ≥16 mm day of trigger (n)	4.13 ± 2.96	0.50 (0.42, 0.58)	<0.001
Number of follicles ≥14 mm day of trigger (n)	2.16 ± 2.23	0.58 (0.47, 0.70)	<0.001
Retrieved oocytes (n)	5.75 ± 4.08	0.45 (0.40, 0.50)	<0.001
Retrieved MII oocytes (n)	5.47 ± 3.93	0.48 (0.43, 0.53)	<0.001
Oocytes normally fertilized of IVF (n)	3.19 ± 3.57	0.52 (0.46, 0.57)	<0.001
Oocytes normally fertilized of ICSI (n)	0.94 ± 2.21	0.16 (0.03, 0.29)	0.014

Correlation analysis between AMH and the number of high-quality embryos

Three models (non-adjusted model, minimally-adjusted model, and fully-adjusted model) were used to evaluate the correlation between AMH and the number of high-quality embryos. The results are shown in Table 3: In non-adjusted model, AMH is positively correlated with the number of high-quality embryos (β = 0.4, 95% confidence interval (CI): 0.3-0.5, P<0.001); in the minimally-adjusted model (adjusted for female age) and fully-adjusted model (adjusted for female age, AFC and COH protocol), the results did not change significantly (β = 0.4, 95% CI: 0.3-0.5, P<0.001, and β = 0.3, 95%CI: 0.2-0.4, P<0.001). To perform sensitivity analysis, AMH was classified into three categories according to the clinical cut-off points. The results showed that the number of high-quality embryos in the high AMH group was 2.2 (95%CI: 0.8-3.7, P=0.003), which was significantly higher than that in the low AMH group (control group) (P=0.005).

Table 3

The relationship between AMH and the number of high-quality embryos in different models
Variable	Non-adjusted model (β, 95%CI, P)	Minimally adjusted model (β, 95%CI, P)	Fully adjusted model (β, 95%CI, P)
AMH (ng/mL)	0.4 (0.3, 0.5) <0.001	0.4 (0.3, 0.5) <0.001	0.3 (0.2, 0.4) <0.001
AMH (ng/mL) (quartile)
<0.7	Ref	Ref	Ref
>=0.7, <8.5	1.7 (0.8, 2.6) <0.001	1.7 (0.8, 2.6) <0.001	0.8 (-0.1, 1.7) 0.066
>=8.5	4.7 (3.3, 6.0) <0.001	4.3 (2.9, 5.6) <0.001	2.2 (0.8, 3.7) 0.003
P for trend	2.2 (1.6, 2.9) <0.001	2.1 (1.4, 2.7) <0.001	1.1 (0.3, 1.8) 0.005
Non-adjusted model: no covariant was adjusted
Minimally adjusted model: Female age (years) was adjusted
Fully adjusted model: Female age (years), AFC (n), COH protocol were adjusted
CI confidence interval, Ref reference

Non-linear Relationship Analysis

The R-based Generalized Additive Model (GAM) was used for nonlinear relationship analysis. The results are shown in Figure 2: the relationship between AMH and the number of high-quality embryos is non-linear (adjusted for female age, AFC and COH protocol). The two-piecewise regression model identified the inflection point of AMH as 7.1 ng/mL. On the left side of the inflection point (AMH<7.1 ng/mL), AMH is positively correlated with the number of high-quality embryos (β = 0.5, 95%CI: 0.3-0.7, P<0.001); and on the right side of the inflection point (AMH≥7.1 ng/mL), there is no correlation between AMH and the number of high-quality embryos (β = 0.1, 95% CI: -0.1-0.2, P=0.518) (Table 4).

Table 4

Two-piecewise linear regression model
Inflection point of AMH	Effect size (β)	95%CI	P value
<7.1	0.5	(0.3, 0.7)	<0.001
>=7.1	0.1	(-0.1, 0.2)	0.518
Effect: AMH Cause: High-quality embryos
Adjusted: Female age (years), AFC (n), COH protocol

Stratification Analysis

The female age, COH protocol and AFC were stratified, and the interaction test showed that the relationship between AMH and the high-quality embryo number was not statistically different among different subgroups (interaction P=0.53, 0.51 and 0.7) (Table 5).

Table 5

The effect of AMH on the number of high-quality embryos among different subgroups
Characteristic	No of participants	Effect size(95%CI)	P for interaction
Female age (years)			0.53
<35	194	0.2 (0.1, 0.4)
>=35,༜38	52	0.4 (0.0, 0.8)
>=38	59	0.5 (0.2, 0.7)
COH protocols			0.51
GnRH-A	147	0.3 (0.1, 0.4)
Mild/PPOS	98	0.4 (0.1, 0.6)
Ultra-Short GnRH agonist	60	0.3 (0.0, 0.7)
AFC (n)			0.7
<6	55	0.6 (0.2, 0.9)
>=6	250	0.3 (0.2, 0.4)
Note 1: The above model was adjusted for Female age (years), AFC (n), and COH protocol

The women from 30 to 44 years old are at high-risk of ovarian aging, and they are the main population receiving ART [5]. The clinical treatment of these patients generally adopts an "empirical" strategy, in which the choice of COH protocol is based on the patient's age, BMI, ovarian reserve, and clinical experience [23]. Before the implementation of ART, clinicians can predict the patient's ovarian response according to age-related ovarian reserve and provide patients with pre-treatment consultation.

In the test of assessing ovarian reserve function, AMH has extremely high specificity and sensitivity [24], and is negatively correlated with age [8]. It can reliably predict the ovarian response in ART and the number of retrieved oocytes [25]. Due to the stability of AMH within and between cycles, as well as the convenience of AMH test, AMH is more and more recognized as the preferred clinical biomarker for the ovarian response to controlled ovarian stimulation [26, 27]. Although AMH testing lacks international standards and there are differences between different testing methods [28], the AMH tests involved in this study all used the same method, which did not affect the reliability of the results.

This retrospective cohort study aims to explore the relationship between AMH and the number of high-quality embryos in the study population. The results showed that younger female, lower bFSH, and higher AFC were associated with higher AMH, which was consistent with the conclusion that AMH was negatively correlated with age and bFSH, and positively correlated with AFC [29]. With the decrease of AMH, the number of retrieved oocytes, the number of MII oocytes, and the number of high-quality embryos decreased significantly, indicating that in this study population, AMH was not only related to the number of retrieved oocytes [30], but also related to the quality of oocytes and embryo development [25]. In both the minimally-adjusted model and the fully-adjusted model, AMH was positively correlated with the number of high-quality embryos (β = 0.4, 95%CI: 0.3-0.5, P<0.001, and β = 0.3, 95%CI: 0.2-0.4, P <0.001). We also treated AMH as a categorical variable according to the clinical cut-off points to divide the patients into low, medium and high AMH groups. The results showed that the high AMH group (≥8.5 ng/mL) had significantly more high-quality embryos than the low AMH group (<0.7 ng/mL) (P<0.05). Sensitivity analysis showed that, compared with the low AMH group, the number of high-quality embryos in high AMH group increased by 2.2 units for each increase in AMH, and the increase was significant (P=0.003). These results indicate that AMH is closely related to the prognosis of reproduction.

The study from Tian Haiqing et al. [31] showed that AMH was positively correlated with the number of available embryos in POSEIDON groups 3 and 4 patients. This study emphasized the number of embryos due to the restriction of poor ovarian reserve (POR) population; the ART outcome was the number of embryos, so embryo quality was not studied. The study population included in this study was 30-44 year-old patients. Our results showed that AMH was positively correlated with the number of high-quality embryos, indicating that AMH was related to both the number and quality of embryos. In addition, the above study did not conduct non-linear relationship analysis, threshold effect analysis, or stratification analysis. In our study, we performed generalized additive model (GAM) analysis, and the results showed that there was a nonlinear relationship between AMH and the number of high-quality embryos. The two-piecewise regression model identified the inflection point as AMH=7.1 ng/mL. On the left side of the inflection point (AMH<7.1 ng/mL), AMH was positively correlated with the number of high-quality embryos (β = 0.5, 95%CI: 0.3-0.7, P<0.001); on the right side of the inflection point (AMH≥7.1 ng/mL), the correlation was not significant (β = 0.1, 95%CI: -0.1-0.2, P=0.518). This result demonstrated that the relationship between AMH and high-quality embryo number was not simply linear, but had obvious segmentation effects. The results of stratified analysis and interaction test showed that, after adjusting for confounding factors (female age, COH protocol, AFC), the positive correlation between AMH and the number of high-quality embryos was stable in each subgroup.

AMH is closely related to female age. Several studies [32–34] measured the low AMH values for certain ages: 3.0 ng/mL for 25 years old, 2.5 ng/mL for 30 years old, 1.5 ng/mL for 35 years old, 1 ng/mL for 40 years old, and 0.5 ng/mL for 45 years old. Since AMH is positively correlated with the number of high-quality embryos, for patients with low AMH values, the clinicians should be optimistic about the assessment of their existing ovarian function and the possible number of high-quality embryos in IVF/ICSI. Embryo quality is an important guideline for successful implantation and live birth during the embryo transfer cycle [35]; it is also a new factor influencing the adverse outcome of newborn [36]. Poor embryo quality is associated with an increased risk of miscarriage, ectopic pregnancy, and low birth weight [37]. On the contrary, high-quality embryos will increase the implantation rate, clinical pregnancy rate, and live birth rate [38]. Therefore, embryo quality is an important surrogate indicator of ART outcome [39].

This study has certain clinical values. First, after adjusting the female age, COH protocol, and AFC, we observed a nonlinear relationship between AMH and the number of high-quality embryos in the first ART cycle of 30-44 year-old infertile women. Secondly, through the threshold effect analysis, we accurately identified the inflection point of the relationship between AMH and high-quality embryo number. Based on these results, the clinicians can obtain information about the potential reproductive prognosis of the patients, which can help estimate the number of IVF/ICSI cycles needed to get a better outcome.

The advantages of this study are as follows: First, the generalized linear model was used to evaluate the linear relationship between AMH and the number of high-quality embryos, and the generalized additive model (GAM) was used to analyze the non-linear relationship; the threshold effect analysis accurately identified the inflection point and provided reliable information for clinical prediction about the potential reproductive prognosis of patients. Secondly, due to the nature of retrospective study, the confounding factors cannot be avoided; however, our study used strict statistical adjustments to minimize the influence of confounding factors on the relationship between exposure factors and outcome variables. Lastly, we used stratified analysis to assess whether the relationship between AMH and high-quality embryo number is consistent between different subgroups, which improved the reliability of the conclusion. The results showed that there was a non-linear correlation between AMH and the number of high-quality embryos, and AMH=7.1 ng/mL was the inflection point. Moreover, the relationship between the two was stable in different subgroups.

The limitation of this study lies in the nature of retrospective study. The results of this study were obtained from the 30-44 year-old patients who used GnRH-A, Mild /PPOS and Ultra-Short GnRH agonist as the first ovarian stimulation protocol; hence, the results cannot be extrapolated to other populations. In addition, this study is a single-center study; thus, a multi-center prospective study is needed to verify the results.

In summary, there is a non-linear correlation between AMH and the number of high-quality embryos in this study population. When AMH<7.1 ng/mL, AMH is positively correlated with the number of high-quality embryos, and this relationship is stable in different stratified variables. Clinically, AMH can be used to predict the potential reproductive prognosis of patients and provide more useful information before ART, thus facilitating the optimization of IVF/ICSI treatment cycle.

Ethics approval

The study received ethical approval from the Institutional Review Board and the need for informed patient consent was waived.

Consent for publication

Not applicable.

Availability of data and material

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Conflicts of interest/Competing interests

The authors declare that they have no conflict of interest.

Funding

This study has received funding by Basic Science Research Program of Nantong (JC2019017).

Authors' contributions

Xiaoling Gu: Data analysis, Manuscript writing

Yannan Chen: Data collection

Minyan Yu: Data collection

Shuping Zhong: Data collection

Xia Wang: Project development

Consent to participate

Not applicable.

Code availability

Not applicable.

Acknowledgements

None.

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Investigating the correlation between AMH and the number of high-quality embryos from IVF/ICSI in 30-44 year-old infertile women

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Abstract

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Background

Materials And Methods

1. Study population and data collection

2. COH protocol [18]

3. Embryo morphology assessment

4. Serological analysis

5. Statistical Analysis

Results

Patient baseline characteristics

Univariate Analysis

Non-linear Relationship Analysis

Stratification Analysis

Discussion

Conclusions

Declarations

References

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