Correlation between the number of oocytes and the increase of polyspermy rate in IVF cycles

Abstract

Aims: To investigate the association between the number of oocytes and the polyspermy rate following in vitro fertilization (IVF) treatment. Materials and methods: 376 IVF cycles with gonadotropin-releasing hormone (GnRH) antagonist protocol in the reproductive center of our hospital were retrospectively included in the analysis, which were divided into five groups according to the number of oocytes retrieved. Group A (78 cases):1–5 oocytes, group B (118 cases): 6–10 oocytes, group C (94 cases): 11–15 oocytes, group D (55 cases): 16–20 oocytes, group E (31 cases): ≥21 oocytes. According to polyspermy rate, 376 IVF cycles were then divided into two groups. Normal level polyspermy group (170 cases): polyspermy rate<6%, and high level polyspermy group (206 cases): polyspermy rate ≥ 6%. The variables with p < .10 in univariate analysis were incorporated into the multiple logistic regression model to control the confounding, and the effect of the number of oocytes on the increase of polyspermy rate was analyzed. Results: Multiple logistic regression analysis showed that after adjustment for confounding factor, the increase risk of polyspermy rate in group B, C, D and E was 1.763, 3.804, 2.021 and 3.208 times of that in group A respectively (OR = 1.763, p = .085; OR = 3.804, p = .001; OR = 2.021, p = .158; OR = 3.208, p = .068, respectively). Conclusion: This result demonstrated that when the oocyte number is 15 or less, the more the oocyte number, the greater the increase risk of polyspermy rate. While, there appears to be little increase risk of polyspermy rate when the oocyte number is more than 15.

Keywords:

• Gonadotropin-releasing hormone (GnRH) antagonist
• in vitro fertilization (IVF)
• polyspermy
• logistic regression

Introduction

Polyspermy refers to the phenomenon that two or more than two sperm enter into oocytes, and form three or more pronucleus. Tt is common abnormal fertilization way in in vitro fertilization (IVF), polyspermy rate of which can reach more than 10%. The embryos from polyspermy oocytes cannot be used for transplantation. High three pro-nuclei (3PN) zygotes proportion associated with normal embryo multinucleation at the two-cell stage [1]. Previous studies had also shown that polyspermy may lead to adverse laboratory and clinical outcomes [2–6]. Therefore, polyspermy rate has become one of the quality control indexes in reproductive laboratories. Vienna Consensus in 2017 [7] recommend that the polyspermy rate in IVF should be limited to 6%. However, despite of strict laboratory standardized procedures, polyspermy rate could still exceed 6% in IVF.

Controlled ovarian hyperstimulation (COH) is an important step in assisted reproductive technology. COH can obtain an appropriate quantity and quality of oocytes, which is the key link of pregnancy [8]. Ovarian reactivity is the sensitivity of ovary to the effect of exogenous gonadotropin (Gn) during COH, and the reactivity determines whether the appropriate number of oocytes can be recruited, which is one of the key factors for the success of COH, and directly affects the process of ovulation induction and the outcome of assisted reproduction [9]. If the ovary did not respond well to Gn stimulation, the number of oocytes was low. On the contrary, if the ovary was extremely sensitive to Gn stimulation, which exceeded the expected level, an excessive number of oocytes were obtained. In the process of COH, different patients have different reactions to Gn, resulting in uneven quality of oocytes which is an important factor affecting polyspermy [10,11].

Previous studies have shown that the more the number of oocytes, the higher the risk to arise polyspermy in IVF cycles [12,13]. However, the effect of the number of oocytes on the increase of polyspermy rate in IVF cycles has not been further analyzed. In this paper, 376 IVF cycles in our center from October 2017 to April 2022 were retrospectively analyzed, aiming to explore the correlation between the number of oocytes and the increase of polyspermy rate in IVF cycles.

Materials and methods

Objects

This study was a retrospective cohort study. From October 2019 to April 2022, 376 IVF patients who were treated with GnRH-ant protocol for COH in the reproductive center of our hospital were selected as the research objects. According to the number of oocytes, patients who met the inclusion criteria were divided into five groups: Group A (78 cases): the number of oocytes ≤ 5, group B (118 cases): 6 ≤ the number of oocytes ≤ 10, group C (94 cases): 11 ≤ the number of oocytes ≤ 15, group D (55 cases): 16 ≤ the number of oocytes ≤ 20, group E (31 cases): the number of oocytes ≥ 21. According to polyspermy rate, 376 IVF cycles were then divided into two groups. Then, following the Vienna Consensus recommendation in 2017 [7], 376 IVF cycles were divided into two groups according to polyspermy rate. Normal level polyspermy group (170 cases): polyspermy rate<6%, and high level polyspermy group (206 cases): polyspermy rate ≥ 6%.

Inclusion criteria: (1) The GnRH-ant protocol was used for COH in this cycle; (2) IVF cycles in our center. Exclusion criteria: (1) Rescue ICSI; (2) Missing of important statistical data. This study was reviewed and approved by the Ethics Committee of Yuncheng Central Hospital (YXLL2022005).

Methods

Detection of sex hormone

Venous blood samples were collected during 8:00–9:00 a.m. on the 2nd to 3rd day of the natural menstrual cycle and on the trigger day of controlled ovulation hyperstimulation (COH) respectively for detection of female basic sex hormones (FSH/LH//E₂/P). Siemens automatic chemiluminescence immunoanalyzer (ADVIA Centaur CP) was used for detection, and direct chemiluminescence immunoassay kit was provided by Siemens Medical Diagnostic Products (Shanghai) Co., LTD.

Controlled ovulation hyperstimulation (COH)

On the 2nd to 3rd day of the natural menstrual cycle, patients were injected 150–300 IU of recombinant human follicle-stimulating hormone (Guonafine, Merck Serono, Germany) or urine gonadotrophin (HMG, Zhuhai Lizhu Pharmaceutical co., LTD., China). On the 6th day of the natural menstrual cycle, patients were given Cetrorelix Acetate Powder for Injection (Cetrotide, Baxter Oncology GmbH, Germany) 0.25 mg/d until the trigger day. When the diameter of one dominant follicle was ≥20mm, the diameters of two to three follicles were ≥18 mm, or the number of follicles of more than 16 mm diameter were exceeded 2/3, 6000–10,000 IU of human chorionic gonadotropin (HCG, Zhuhai Lizhu Pharmaceutical co., LTD., China) was injected, and 36h later, oocytes were harvested by ultrasound-guided vaginal puncture.

Semen treatment and short-term insemination

Patients were required to abstain from sex for 3–5 days before oocytes collection. On the day of oocytes collection, patients were required to masturbate for semen collection. Density gradient centrifugation and upstream method were used to process semen. Density gradient centrifugal separation liquid was Spermient 40% and 80% (COOK, Australia). The washing solution was gamete buffer (GB, COOK, Australia), and the upstream solution was fertilization medium (FM, COOK, Australia). The four-well method was used for insemination. Before the oocytes were collected, 0.6 ml of FM was added to each well of four-well dish, covered with oil, and used after balancing overnight. 40 h after HCG injection, the mixed sperm supernatant was added into the four-well dish at the density of 50,000 sperm per oocyte. After sperm motility and density were confirmed under the microscope, oocytes were added into the four-well plate, with 2–3 oocytes per well, and degranulation was performed 4 h later.

Denuding and fertilization observation

4 h after IVF insemination, denuding pipette (ID:140um, COOK, Australia) was used for degranulation. The oocytes were then transferred to overnight-balanced cleavage medium (CM, COOK, Australia), and the discharge of the second polar body was observed under the microscope. When the number of oocytes of two polar bodies is greater than or equal to 1/2 of the number of mature oocytes, the oocytes were put back into the incubator (37 °C, 6%CO₂) for further culture, and the fertilization situation was observed 18 h later. A zygote with three or more pronucleus (PN)18 h after fertilization was considered as polyspermy. IVF polyspermy rate = > 2PN oocyte number/all oocyte number × 100% [7].

Index of observation

The basic data of patients included female age, duration of infertility, type of infertility, infertility factors, body mass index (BMI) and AMH. The included clinical data included FSH days, total FSH amount, HMG days, total HMG amount, Gn starting dose, serum LH/E₂/P levels on trigger day, the number of oocytes, the proportion of immature oocytes and the level of polyspermy rate in five oocyte groups.

Statistical analysis

SPSS 26.0 software was used for statistical analysis. The schapiro-Welk (K-W) method was used to test the normality of measurement data. Because of non-normal distribution, the measurement data were expressed as the median (25th percentile, 75th percentile) M(P25, P75), differences in oocyte groups were performed using the Kruscarl-Wallis H(K) test. The count data were expressed by composition ratio, Pearson chi-square test was used for comparison in groups. Multivariate logistic regression analysis was used to evaluate the effect of the number of oocytes on polyspermy rate in IVF. The confounding factors were tested by univariate logistic regression analysis, and then corrected by multivariate logistic regression model. The variables with p < .10 in univariate analysis (infertility factors, AMH, FSH days, Gn starting dose, E₂ level on trigger day, P level on trigger day, the number of oocytes and proportion of immature oocytes) were incorporated into the multiple logistic regression model to analysis the effect of the number of oocytes on polyspermy rate in IVF. Using A group as the control group, Odds ratios (ORs) and 95% confidence intervals (CI) were calculated before and after polyspermy rate adjustment. All tests were two-sided, and p < .05 was considered statistically significant.

Results

Overall data

From October 2019 to April 2022, the number of IVF cycles using GnRH-ant protocol for COH in the reproductive center of our hospital was 452. Excluding 31 cycles of rescue ICSI, 45 cycles of missing important statistical data, 376 IVF cycles were included in this study.

Basic patient characteristics and clinical data

Female age, infertility factors, AMH, FSH total dose, HMG days, HMG total dose, Gn starting dose, LH level on trigger day, E₂ level on trigger day, P level on trigger day, number of oocytes, polyspermy rate, and level of polyspermy rate in five oocyte groups were different significantly (p<.05). Other data had no significant difference (p > 0.05), as shown in Table 1.

Table 1. Basic patient characteristics and clinical data[M(P25, P75), n(%)].

Factors	A group (n = 78)	B group (n = 118)	C group (n = 94)	D group (n = 55)	E group (n = 31)	z (χ^2)	P
Female age	32.00 (29.00,35.00)	31.00 (27.00,34.00)	30.00 (27.00, 32.00)	28.00 (26.00, 31.00)	28.00 (26.00, 30.25)	36.624	0.000*
Infertility years	3 (2,5.25)	3 (2,5)	3 (2,5.25)	3 (2,4)	3.5 (2,5.25)	0.493	0.974
Infertility type (primary/secondary)	34/44	53/65	45/49	29/26	21/10	6.363	0.174
Infertility factors(Pelvic and fallopian tube/Ovulatory dysfunction/others)	45/13/20	74/20/24	69/17/8	37/16/2	19/9/3	22.487	0.004*
Body mass index (BMI, kg/m^2)	23.15(20.98, 26.20)	22.60(20.70, 25.20)	23.95(21.50, 27.03)	22.50(21.00,25.50)	24.30(21.80,26.43)	7.627	0.106
AMH	1.14(0.70, 1.94)	1.63(1.12, 2.90)	2.38(1.62, 3.98)	5.58(2.46,10.79)	6.11(3.98,9.93)	138.391	0.000*
FSH days	9.5(8, 11)	10(9, 11)	10(9, 11)	10(9, 11)	10(9, 11)	6.564	0.161
FSH total dose (U)	2100.00(1781.25,2400.00)	2175.00(1800.00,2625.00)	1950.00(1650.00,2325.00)	1425.00(1350.00,1950.00)	1462.50(1275.00,1950.00)	50.410	0.000*
HMG days	7(4,10)	5(3, 8)	5(2, 9)	2(1,6)	1.5(0,3.5)	40.681	0.000*
HMG total dose (U)	825(506.25, 1368.75)	600(300, 975)	525(225,975)	225(75,750)	150(0,393.75)	52.374	0.000*
Gn starting dose (U)	300(225, 300)	300(225, 300)	225(150,300)	150(150,225)	150(150,225)	119.238	0.000*
LH level on trigger day (U/L)	2.05(1.58,3.51)	1.91(1.27, 3.41)	1.65(0.87,2.70)	1.72(1.00,2.94)	1.98(1.08,4.35)	11.844	0.019*
E2 level on trigger day(pg/ml)	1538.62(1101.95,2255.32)	2786.93(1994.57,3620.50)	3652.80(2957.21,4691.44)	4960,68(4023.48,6763.48)	7612.67(4907.16,10747.88)	199.046	0.000*
P level on trigger day ((ng/ml))	0.76(0.40, 1.14)	0.99(0.68, 1.46)	1.17(0.97,1.62)	1.34(1.04,1.83)	1.77(1.23,2.45)	74.597	0.000*
Number of oocytes	4(3,5)	8(7,9)	12(11,14)	17(16,19)	26(24,30)	353.863	0.000*
Proportion of immature oocytes, n (%)	0.00(0.00,33.33)	11.11(0.00,25.00)	9.09(6.67,25.00)	12.50(0.00,22.22)	12.50(4.41,21.99)	2.827	0.587
Level of polyspermy rate [Normal level(polyspermy rate<6%)/High level(polyspermy rate ≥ 6%)]	52/26	58/60	29/65	22/33	9/22	26.940	0.000*

注:*p < .05.

Logistic regression analysis of factors related to increase of polyspermy rate in IVF

Univariate logistic regression analysis showed that infertility factors, AMH, FSH days, Gn starting dose, E₂ level on trigger day, P level on trigger day, the number of oocytes and the proportion of immature oocytes were possible effecting factors of polyspermy rate (p<.10). Multiple logistic regression analysis showed that the increase risk of polyspermy rate in group B, C, D and E was 1.763, 3.804, 2.021 and 3.208 times of that in group A respectively (OR = 1.763, p = .085; OR = 3.804, p = .001; OR = 2.021, p = .158; OR = 3.208, p = .068, respectively). As shown in Table 2.

Table 2. Logistic regression analysis of factors related to polyspermy rate of IVF cycles.

Factors	Univariate regression			Multiple regression
	P value	OR	95%CI	P value	OR	95%CI
Female age	0.682	0.991	0.947-1.037
Infertility years	0.783	1.010	0.942-1.083
Infertility type (secondary vs. primary)	0.574	1.124	0.748-1.687
Infertility factors	0.036*			0.197
Pelvic and fallopian tube factors		1.000			1.000
Ovulatory dysfunction	0.283	0.752	0.447-1.265	0.232	0.701	0.391-1.255
Others	0.012*	0.470	0.261-0.845	0.123	0.600	0.313-1.149
Body mass index (BMI, kg/m^2)	0.767	1.008	0.955-1.065
AMH	0.028*	1.066	1.007-1.128	0.800	1.011	0.930-1.098
FSH days	0.034*	1.133	1.010-1.271	0.278	1.074	0.944-1.223
FSH total dose (U)	0.395	1.000	0.999-1.000
HMG days	0.709	0.990	0.939-1.044
HMG total dose (U)	0.579	1.000	1.000-1.000
Gn starting dose (U)	0.040*	0.997	0.994-1.000	0.698	1.001	0.996-1.005
LH level on trigger day (U/L)	0.117	0.921	0.831-1.021
E2 level on trigger day(pg/ml)	0.000*	1.000	1.000-1.000	0.604	1.000	1.000-1.000
P level on trigger day ((ng/ml))	0.003*	1.604	1.172-2.196	0.328	1.218	0.820-1.807
Proportion of immature oocytes	0.000*	0.036	0.009-0.154	0.000*	0.026	0.005-0.131
Number of oocytes	0.000*			0.015*
A group		1.000			1.000
B group	0.016*	2.069	1.143-3.744	0.085	1.763	0.924-3.364
C group	0.000*	4.483	2.357-8.525	0.001*	3.804	1.746-8.285
D group	0.003*	3.000	1.466-6.139	0.158	2.021	0.761-5.368
E group	0.001*	4.889	1.973-12.112	0.068	3.208	0.918-11.210

注:*p < .05.

Discussion

We analyzed the association between the number of oocytes and the increase of polyspermy rate in IVF. Univariate logistic regression analysis showed that infertility factors, AMH, FSH days, Gn starting dose, E₂ and P on trigger day, the number of oocytes and the proportion of immature oocytes may affect polyspermy rate (p < .10). Therefore, these 8 variables were used as confounding factors to correct polyspermy rate in multivariate logistic regression model in this study. The results showed that the number of oocytes was positively correlated with the increase of polyspermy rate in IVF when the oocyte number was 15 or less. While, when the oocyte number was more than 15, there appeared to be no longer elevated risk of polyspermy rate.

At present, several domestic and foreign studies reported the relationship between the number of oocytes and the occurrence of polyspermy in IVF. In 2019, Lu and He [14] divided 1763 IVF/ICSI cycles into 5 groups according to the different number of oocytes: ≤5 (160 cases), 6–10 (397 cases), 11–15 (421 cases), 16–20 (342 cases), ≥21 (443 cases). The results showed that there was no significant difference in the fertilization rate of different oocyte groups. However, they did not clarify the relationship between different number of oocytes and polyspermy. Li et al. [12] conducted a retrospective study with 508 donor in vitro fertilization (IVF-D) cycles using the standard long protocol for ovulation induction. The 508 cycles were divided into normal fertilization group (without polyspermy) and polyspermy group (with polyspermy) according to whether polyspermy occurred in this cycle. The results showed that the number of oocytes in polyspermy cycle was significantly higher than that in normal fertilization cycle. Zhao et al. [13] also divided 686 IVF cycles into polyspermy group (348 cases) and non-polyspermy group (338 cases) according to the presence or absence of polyspermy, which also showed that the higher the number of oocytes, the more possibility of polyspermy presence. However, the above studies only showed the relationship between the number of oocytes and the occurrence of polyspermy, which were not corrected by confounding factors, and the influence and degree of the number of oocytes on the increase of polyspermy rate were not clarified. In our study, after adjusting for infertility factors, AMH, FSH days, Gn starting dose, E₂ and P on the trigger day, and the proportion of immature oocytes, the increase risk of polyspermy rate in group B and C was 1.763 and 3.804 times of that in group A respectively (OR = 1.763, p = .085; OR = 3.804, p = .001, respectively). The increase risk of polyspermy rate in group D and E was 2.021 and 3.208 times of that in group A respectively (OR = 2.021, p = .158; OR = 3.208, p = .068, respectively). While, due to the small sample size in group D (55 cases) and E (31cases), the difference in the increase risk of polyspermy rate between group D/E and group A was not statistically significant.

It is worth noting that Gonadotropin-releasing hormone antagonist (GnRH-ant) protocol which uses GnRH-ant to inhibit the endogenous LH peak in the middle and late follicles [15] is applicable to a wide range of people whose age/ovarian reserve function and others vary greatly, so some basic data and clinical data of patients in different oocytes groups in our study were significantly different. Although confounding factors were used to correct polyspermy rate, the small sample size may affect the statistical efficiency. Moreover, chromosomal and genetic analyses were not performed and the influence of chromosomal abnormalities and genetic mutations on fertilization cannot be excluded. Finally, this study is a single-center retrospective study, which may have the problem of heterogeneity in the study population, and the level of evidence is not as high as that of randomized controlled studies. The conclusion of our study still needs to be further confirmed by a prospective study with a large sample size of multi-center.

The use of large doses of exogenous Gn in the process of COH makes estrogen far beyond the physiological level. The high estrogen status will affect the normal development and regulation of oocytes, reducing the maturation rate of oocytes and leading to the asynchronism of nuclear and cytoplasmic maturation [16]. The more the number of oocytes, the more likely the uneven oocytes quality, and the more likely to occur cytoplasmic incomplete maturation or overmature oocytes, which increases the risk of polyspermy [17]. But the number of oocytes is not simply positively correlated with the increase of polyspermy rate in IVF. Further molecular mechanisms are unknown.

In conclusion, our result demonstrated that oocyte number was one of the independent risk factors for the increase of polyspermy rate in IVF. When the oocyte number is 15 or less, the more the oocyte number, the greater the increase risk of polyspermy rate. It is of great significance to control the number of oocytes to improve the index of polyspermy in laboratory. But when the number of oocytes exceeds 15, the number of oocytes will no longer affect the polyspermy rate.

Disclosure statement

The authors report no conflict of interest.

Additional information

Funding

This work was supported by Scientific Research Project of Shanxi Provincial Health Commission (2022054).