Effect of three pro-nuclei (3PN) incidence on pregnancy outcomes in women with good prognosis undergoing in vitro fertilization with donor sperm (IVFD)

doi:10.21203/rs.3.rs-1732295/v1

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Effect of three pro-nuclei (3PN) incidence on pregnancy outcomes in women with good prognosis undergoing in vitro fertilization with donor sperm (IVFD)

https://doi.org/10.21203/rs.3.rs-1732295/v1

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Purpose

To investigate the factor of three pro-nuclei (3PN) incidence and its effect on pregnancy outcomes in women with good prognosis undergoing in vitro fertilization with donor sperm (IVFD).

Methods

We retrospectively identified all IVFD embryo transfers (IVFD-ETs) for the first time between January 2014 and January 2021 at our hospital. Women aged ≤ 35 years who received the long/ultra-long follicular phase agonist protocol were included. The patients were divided into two groups as follows: 3PN = 0% group included patients with no 3PN zygotes and 3PN > 0% group included patients with 3PN zygotes.

Results

We observed that the number of retrieved oocytes was independently associated with 3PN incidence (OR: 1.078, 95% CI 1.045–1.113, p < 0.001). Both the 3PN = 0% and 3PN > 0% groups showed no significant differences in the cleavage, high-quality embryo and available embryo rates (p > 0.05). Our data indicated that the 3PN = 0% group had significantly higher ongoing pregnancy (68.50 versus 62.76%, p = 0.033) and live birth (66.93 versus 59.84%, p = 0.009) rates compared with that in the 3PN > 0% group. The miscarriage rate was significantly lower in the 3PN = 0% group than that in the 3PN > 0% group (7.78 versus 12.26%, p = 0.026). The multinucleation at two-cell stage (MN2) rate was slightly higher in the 3PN > 0% group than that in the 3PN = 0% group (23.57 versus 16.11%, p = 0.085).

Conclusions

The incidence of 3PN zygotes made negative influence on the pregnancy outcomes in women with good prognosis undergoing IVFD-ETs. 3PN incidence might be associated with normal embryo multinucleation at two-cell stage.

3PN

IVF

Donor sperm

Pregnancy outcomes

Infertility affected nearly 15% of couples worldwide. 20–30% of infertile cases were due to a male factor, 20–35% to a female factor, and 25–40% of infertile cases were due to both factors. There was a gradual rise in the requirement of fertility treatments using donor sperm in recent years [1].

A variety of polyspermy-preventing mechanisms existed to ensure the incidence of normal fertilization. It was well known that two mechanisms (“oocyte membrane block” and “zona reaction”) were significant to prevent polyspermy which existed in marine animals and mammals, including human. Nevertheless, three pro-nuclei (3PN) zygotes still occurred in some patients during the process of conventional in vitro fertilization (C-IVF). It was reported that the 3PN rate was 2–10% during the process of C-IVF [2].

It was confirmed that the incidence of 3PN zygotes was associated with a high response to gonadotropin stimulation which was manifested as an increase in the number of oocytes retrieved [3]. Thus, more immature and aging oocytes were produced proportionately and the appearance of 3PN zygotes might result from these poor oocytes. Both sperm- and oocyte-derived factors were significant to ensure the achievement of normal fertilization. Unlike the paternal genome, the maternal genome carried a heavy footprint of parental aging. There was no doubt that the oocyte was the major determinant of embryo developmental competence [4].

The way a single embryo was affected might indicate how the whole cohort was affected, even if the other embryos did not show the same characteristics. Therefore, we aimed to investigate the effect of 3PN incidence on pregnancy outcomes in women with good prognosis undergoing IVFD-ETs.

Study design and patients

This study included 1623 fresh IVFD-ETs for the first time between January 2014 and January 2021 at our hospital. After excluding subjects with in-vitro maturation, previous uterine or ovary surgery and uterine malformation, a total of 1587 women were included during the study period. All female patients were not beyond 35 years old to eliminate possible age-related cycle characteristics, and only the women receiving long/ultra-long agonist protocol were included in the analysis. Ultimately, 1250 patients were included and divided into two groups as follows: 3PN=0% group included patients with no 3PN zygotes (n=635) and 3PN>0% group included patients with 3PN zygotes (n=615).

Ovarian stimulation protocol

All first-attempt down-regulated ovarian stimulation cycles were long or ultra-long protocols with GnRH agonist (GnRH-a, Decapeptyl, Germany) and recombinant FSH (GONAL-f, Merck, Serono, Italy; Purge, Organon, the Netherlands) for controlled ovarian hyperstimulation (COH). The details of the ovarian stimulation protocol for the ovarian stimulation regimen were as previously described [5].

Sperm preparation and IVFD procedure

The donor sperm samples were supplied by Shaanxi Province Human Sperm Bank. The general procedures for donor recruitment, sperm freezing, and selection of recipients were the same, in accordance with the standards of National Health and Family Planning Commission (NHFPC) of the People’s Republic of China. All donor samples were studied by means of periodical analysis to eliminate the presence of sexually transmitted diseases and genetic disorders. Briefly, the requirements for donor semen are as follows: semen volume ≥2 ml, concentration ≥60 × 10⁶/ml, progressive motility ≥60%, and normal morphology of ≥30%, and patients were healthy and had no chromosome abnormality [6]. Samples were thawed and prepared by density gradients when required, after selection to match the receiving couple’s phenotypical characteristics and blood type.

Conventional IVF fertilization was performed 2-2.5 h after oocyte retrieval. Each oocyte was incubated with approximately 40,000 sperm and fertilization was allowed to occur naturally.Generally, two eggs were co-cultured with sperms in each well.

Embryo culture and assessment with conventional morphology

The OCCs were cultured in the medium (IVF; Vitrolife, Sweden) after retrieval. The zygotes were shifted to the cleavage medium (G-1; Vitrolife) 5 hours after the fertilization of IVF. The embryos to culture blastocyst on day 3 were transferred to the blastocyst medium (G-2; Vitrolife) until day 6. All medium were covered with paraffin oil in a humidified atmosphere at 37°C for prior 24 h.

Embryo morphology was assessed by two well-trained embryologists on the mornings of days three and five of embryo development using an inverted microscope (Eclipse TE 300; Nikon, Tokyo, Japan) under 400 × magnification. A morphologic score was given for day-3 and day-5 embryo according to using published criteria [7,8].

Observation of multinucleation at two-cell stage (MN2)

For culture with the EmbryoScope (Vitrolife, Sweden), dedicated 16-well plates were prepared with 25 ml microdrops of culture medium (Vitrolife, Sweden) overlaid by 1.4 ml of mineral oil (Vitrolife) at 37°C, in 6% CO₂, 5% O₂, and 89% N₂. Images of each embryo were analyzed retrospectively using the EmbryoViewer external image analysis software (Unisense FertiliTech, Sweden). Images were acquired every 20 min in seven different focal planes during culture. The embryos with MN2 were recorded by using time-lapse monitoring.

ET and pregnancy confirmation

The ET catheter (Cook Ireland Ltd., Limerick, Ireland) was used for transfer. Before transfer, any vaginal and cervical secretions were gently removed from the vagina/cervix with small pledgets of cotton wool, moistened with warm normal saline. The mucus in the cervical canal was wiped away. After transfer, the catheter was checked for retained embryos and the presence of blood. After ET, all patients were given luteal support (Duphaston; progesterone injection).

Serum β-hCG was measured 14 days after cleavage-stage embryo transfer and 9 days after blastocyst transfer. Clinical pregnancy was defined by the ultrasound confirmation of an intrauterine gestational sac after 6 weeks of gestation. Ongoing pregnancy was defined as fetal cardiac activity at 12 weeks. Live birth was defined as the delivery of a live-born infant (> 24 weeks of gestation).

Statistical analysis

Comparison of the results between groups in the case of continuous variables was made by using Student’s t test for data with normal distribution and non-parametric Mann-Whitney U-test for data with skewed distribution. Comparison of the results between groups in the case of categorical variables was expressed as number and percentage and analyzed using the Chi-square test or Fisher exact test. Forward logistic regression analysis was performed to determine the risk factors for 3PN incidence. The statistical analysis was performed with SPSS version 21 (IBM Corp., USA). Differences were considered statistically significant when p < 0.05.

The study flow chart was shown in Fig. 1. We observed that the proportion of cycles with 3PN zygotes was 50.80% in women with good prognosis undergoing IVFD-ETs (Fig. 2). Potential factors entered as independent variables in the multivariate logistic regression analysis for 3PN incidence included age, BMI, basal FSH, basal E₂, total Gn dosage, Gn stimulation time and the number of retrieved oocytes. We observed that the number of retrieved oocytes was independently associated with 3PN incidence (Table 1).

Table 1

Multivariate logistic regression analysis of female risk factors for 3PN incidence.
	OR	95% CI	p value
Age, years	0.972	0.937–1.010	.143
BMI, kg/m²	0.979	0.940–1.019	.302
Basal FSH (mIU/ml)	0.981	0.929–1.035	.477
Basal E₂ (pg/ml)	0.999	0.994–1.004	.782
Total Gn dosage (IU)	1.000	1.000–1.000	.950
Gn stimulation, days	1.009	0.980–1.038	.561
No. of retrieved oocytes (n)	1.078	1.045–1.113	< .001
CI: confidence interval; OR: odds ratio; BMI: body mass index; FSH: follicle-stimulating hormone; E₂: estradiol; Gn: gonadotropin; HCG: human chorionic gonadotropin.

The distribution of cycle-specific parameters between groups was described in Table 2, and no significant differences were observed in female’s age, BMI, basal FSH, basal E₂, total Gn dosage, Gn stimulation time and endometrial thickness between the 3PN = 0% and 3PN > 0% groups. The number of retrieved oocytes was significantly higher in the 3PN > 0% group than that in the 3PN = 0% group (p < 0.001). We observed no significant differences in cleavage (97.10 versus 96.63%, p = 0.205), high-quality embryo (56.65 versus 56.63%, p = 0.979) and available embryo (86.92 versus 88.24%, p = 0.061) rates between the 3PN = 0% and 3PN > 0% groups (Table 2).

Table 2

Characteristics of study population and laboratory data.
	3PN = 0%	3PN > 0%	p value
No. transfers	635	615	/
Age, years	28.27 ± 3.09	27.93 ± 3.13	.053
BMI, kg/m²	22.15 ± 3.04	22.02 ± 2.98	.217
Basal FSH (mIU/ml)	6.87 ± 2.55	6.67 ± 1.83	.110
Basal E₂ (pg/ml)	46.13 ± 24.67	46.05 ± 22.29	.947
Total Gn dosage (IU)	2081.80 ± 782.32	2024.37 ± 707.53	.185
Gn stimulation, days	10.50 ± 2.36	10.79 ± 7.26	.348
No. of retrieved oocytes (n)	10.19 ± 4.24*	11.87 ± 4.20*	< .001
Infertility type (%,n )			.699
Primary infertility	77.80 (494)	78.70 (484)
Secondary infertility	22.20 (141)	21.30 (131)
Endometrial thickness (mm)	12.05 ± 2.57	12.08 ± 2.53	.626
No. transferred embryos (%,n )			.520
1	45.83 (291)	47.64 (293)
2	54.17 (344)	52.36 (322)
Cleavage- or blastocyst-stage transfers (%,n )			.727
Cleavage-stage	49.61 (315)	48.62 (299)
Blastocyst-stage	50.39 (320)	51.38 (316)
Normal fertilization (%,n)	69.26 (4481/6470)*	62.87 (4576/7279)*	< .001
Cleavage (%,n )	97.10 (4351/4481)	96.63 (4422/4576)	.205
High-quality embryo (%,n )	56.65 (2465/4351)	56.63 (2504/4422)	.979
Available embryo (%,n )	86.92 (3782/4351)	88.24 (3902/4422)	.061
* was significantly different.

Our data indicated that the 3PN = 0% group had significantly higher ongoing pregnancy (68.50 versus 62.76%, p = 0.033) and live birth (66.93 versus 59.84%, p = 0.009) rates compared with that in the 3PN > 0% group. The miscarriage rate was significantly lower in the 3PN = 0% group than that in the 3PN > 0% group (7.78 versus 12.26%, p = 0.026). For patients with cleavage-stage embryo transfers, we observed no significant differences in implantation, clinical pregnancy, ongoing pregnancy, twin pregnancies, ectopic pregnancy, miscarriage and live birth rates between the 3PN = 0% and 3PN > 0% groups (p > 0.05). For patients with blastocyst-stage embryo transfers, we observed that the live birth rate was significantly decreased in the 3PN > 0% group than that in the 3PN = 0% group (60.13 versus 67.81%, p = 0.044) (Table 3).

Table 3

Pregnancy outcomes of study population.
Parameter	Cleavage-stage		p value	Blastocyst-stage		p value	Total		p value
Parameter	3PN = 0%	3PN > 0%	p value	3PN = 0%	3PN > 0%	p value	3PN = 0%	3PN > 0%	p value
No. transfers	315	299	/	320	316	/	635	615	/
Implantation (%,n)	52.52 (302/575)	50.55 (278/550)	.507	69.31 (280/404)	65.12 (252/387)	.229	59.45 (582/979)	56.56 (530/937)	.201
Clinical pregnancy (%,n )	71.43 (225/315)	67.22 (201/299)	.259	74.38 (238/320)	69.62 (220/316)	.182	72.91 (463/635)	68.46 (421/615)	.083
Ongoing pregnancy (%,n )	67.94 (214/315)	62.21 (186/299)	.137	69.06 (221/320)	63.29 (200/316)	.124	68.50 (435/635)*	62.76 (386/615)*	.033
Twin pregnancies (%,n )	34.22 (77/225)	37.81 (76/201)	.441	17.65 (42/238)	14.55 (32/220)	.368	25.70 (119/463)	25.65 (108/421)	.871
Ectopic pregnancy (%,n )	1.33 (3/225)	0.50 (1/201)	.372	0 (0/238)	0.45 (1/220)	.298	0.65 (3/463)	0.47 (2/421)	.732
Miscarriage (%,n )	7.56 (17/225)	11.44 (23/201)	.170	7.98 (19/238)	13.18 (29/220)	.070	7.78 (36/463)*	12.26 (52/424)*	.026
Live birth (%,n )	66.03 (208/315)	59.53 (178/299)	.096	67.81 (217/320)*	60.13 (190/316)*	.044	66.93 (425/635)*	59.84 (368/615)*	.009

For patient with 3PN zygotes, we observed that transfers at cleavage-stage with double embryos and transfers at blastocyst-stage had similar clinical pregnancy (70.51 versus 69.62%, p > 0.05), ongoing pregnancy (64.54 versus 63.29%, p > 0.05) and live birth (61.35 versus 60.13%, p > 0.05) rates. The twin pregnancies rate was significantly decreased by extended culture to blastocyst-stage for transfer compared with double cleavage-stage embryo transfers (14.55 versus 49.35%, p < 0.05) (Fig. 3). By using time-lapse monitoring, 180 normal embryos from the 3PN = 0% group and 157 normal embryos from the 3PN > 0% group were observed and recorded. The MN2 rate was slightly higher in the 3PN > 0% group than that in the 3PN = 0% group (23.57 versus 16.11%, p = 0.085) (Fig. 4)..

A variety of polyspermy-preventing mechanisms existed to ensure the incidence of normal fertilization [9–11]. Nevertheless, 3PN zygotes still occurred in some patients during the process of C-IVF. The reasons for causing 3PN incidence were complicated which were mainly due to oocyte-derived meiotic failure and polyspermic fertilization. It was reported that 3PN incidence was associated with oocyte quality and sperm abnormality. The donor semen had strict standards in morphology, concentration, motility and chromosome analysis. In this study, the donor semen was selected to exclude abnormal sperm influence on reproductive outcomes as much as possible. Physiologically, patients with strong response to the drug often produced proportionately more immature or overripe oocytes which increased the probability of 3PN incidence. Immature egg could not produce perfect cortical reaction and zona reaction which resulted in the increase of polyspermy rate. After aging of overripe egg, only part or no cortical granules could be released. It resulted in incomplete or unsuccessful cortical reaction which increased the probability of polyspermy incidence.

A recent report indicated that high 3PN zygotes proportion might be associated with the MN2 incidence [12]. In this study, we observed that the MN2 rate was slightly improved in the 3PN > 0% group than that in the 3PN = 0% group by using time-lapse monitoring. Multinucleation was a nuclear abnormality caused by an error in the cytokinesis process [13]. Jackson et al found multinucleation present in cycles that exhibited a more exuberant response to therapy, as indicated by both the higher E₂ levels and large oocyte yields [14]. Rita et al showed that most of the patients with MN2 incidence had a higher number of follicles and retrieved oocytes [15]. Our data indicated that the number of retrieved oocytes was independently associated with 3PN incidence which was consistent with previous report [16]. To sum up, higher peak serum E₂ level and more oocytes retrieved might be associated with the incidence of 3PN zygotes and MN2 embryos.

Figueira et al. observed a 3.5-fold higher risk of miscarriage in cycles with a 3PN incidence of > 25% [17]. Molina et al. indicated that high proportion of 3PN zygotes had a statistically significant inverse effect on clinical pregnancy rate [18]. Rosen et al. showed that 3PN formation in this subset of patients was a significant negative predictor of implantation [19]. Our results also revealed that the patients with no 3PN zygotes had better clinical pregnancy, ongoing pregnancy and live birth rates compared with the patients with 3PN zygotes. Thus, the incidence of 3PN zygotes might prompt that the whole cohort was not optimal even if some embryos showed the normal fertilization characteristics.

For embryo culture with conventional tri-gas incubator, the phenomenon of MN2 was always not observed. Thus, it might increase the probability of transferring normal embryo with MN2. It was reported that the phenomenon of MN2 was associated with decreased blastocyst formation, lower implantation and reduced live birth rates [20,21]. A retrospective analysis of cleavage-stage embryos also indicated that the appearance of MN2 was associated with impaired cleavage and increased fragmentation [22]. Nevertheless, our data showed that there were no significant differences in the pregnancy outcomes for patients with cleavage-stage embryo transfers between the 3PN = 0% and 3PN > 0% groups. The reason might be that the transfers with double cleavage-stage embryos were always performed on day 3.

Egashira et al. demonstrated that the blastocyst from MN2 embryos had similar implantation potential compared with that from normal embryos [23]. Balakier et al. indicated that most MN2 embryos had the capacity for self-correction during early cleavage divisions and could develop into euploid blastocysts resulting in healthy babies [24]. These results suggested that extended culture of MN2 embryos to the blastocyst-stage for transfer might improve pregnancy outcomes. Nevertheless, we observed that the live birth rate was significantly decreased in the 3PN > 0% group than that in the 3PN = 0% group for patients with blastocyst-stage embryo transfers. For better sperm quality and fewer female fertility problems, IVF cycles using donor sperm had better pregnancy outcomes compared with those using partner sperm. Twin pregnancies were more likely to happen in the IVFD-ETs if double blastocyst-stage embryos were transferred. Thus, single blastocyst transfer was always recommended in our center which might be associated with reduced live birth rate in the 3PN > 0% group.

For limited date of IVFD-ETs from the time-lapse monitoring, it was not enough to prove that the frequency of MN2 was associated with the incidence of 3PN zygotes. The poorer pregnancy outcome of patients with 3PN zygotes in the IVFD-ETs was not necessarily attributed to transfers with more MN2 embryos. There was no doubt that a study with large sample size was needed to confirm our observation by time-lapse monitoring.

Author contributions MZL: manuscript writing, XX: data analysis, JZS: protocol development.

Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript

Compliance with ethical standards

Conflicts of interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical statement The study protocol was approved by the Ethics Committee of Northwest Women and Children’s Hospital (No. 2022007). All research was performed in accordance with relevant guidelines and regulations. Patient consent was not required due to the retrospective nature of the study.

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Effect of three pro-nuclei (3PN) incidence on pregnancy outcomes in women with good prognosis undergoing in vitro fertilization with donor sperm (IVFD)

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Abstract

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Introduction

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