The effect of short-term disturbance of day 3 embryo culture on the development and implantation

Abstract

This study is carried out to estimate the effect on embryo development and implantation potential performed by inevitably occurring short-term 3-day embryo culture condition disorders due to light microscopy applied for morphology parameter assay. One thousand two hundred and fifty-three IVF program results were analyzed to measure the pregnancy rate on embryo transfer and the percentage of embryos reached the blastocyst stage. In 58% of cases, on the 3rd day the quality of embryos was not evaluated (n = 730), while in 42% of cases the evaluation was done on day 3 (n = 523). Embryo development estimation on day 3 affects the pregnancy rate and implantation potential especially in patients of older age group. Additional embryo analysis also affects the number of blastocysts obtained. The pregnancy rate for 3rd and 5th day transfer does not differ. Additional analysis of the 3rd day embryos reduces the PR in patients of the older age group. Embryo culture condition variation has a considerable impact on obtained blastocyst number (i.e blastocyst formation) especially in patients of the older age group.

Keywords:

• Infertility
• IVF
• embryo culture
• blastocyst formation
• pregnancy rate

Introduction

IVF obtained embryo culture is the most important stage on the way to pregnancy and healthy childbirth. Nowadays, great attention is paid to this stage quality control, which includes culture condition control: temperature, culture medium pH, osmolarity, etc. [1]. One of the most important criteria for culture quality is embryo implantation frequency.

The present common practice requires embryo cultivation for up to 5 days of development. It has been proved that pregnancy frequency, as well as childbirth, is statistically higher in case of 5th-day transfer, which is determined by a possibility to select best quality embryos, having achieved the blastocyst stage [2–4]. Best strategy of embryo cultivation is to keep them in optimal conditions. Modern incubators along with upgraded culture medium allow controlling most of the parameters without interference. There are two reasons for breaking ‘ideal culture conditions’: necessity to obtain information of fertilization and embryo development and also applying multistage culture medium, which inevitably leads to transferring embryos from one culture medium into another of the next stage. This causes short-term variations of several parameters: pH, temperature, illumination intensity. The listed disorders are risk factors affecting embryo quality and their implantation potential.

The effect of each factor has been already reported. Carbonate buffer is a common pH regulator for any culture medium. Optimal cultivation is performed in gas incubators with 5.5–6% Со₂ content, able to maintain 7.2–7.3 pH level. Even a slight pH increase during manipulations outside the incubator substantially affects the homeostasis (enzyme activity, cell cleavage, differentiation, transporting via membrane, cytoskeleton elements) and gene expression levels [5–7].

One of the most important culture factors is temperature maintaining. Oocyte is most sensible to temperature variations in vitro. Even 1°С temperature move may cause cleavage spindle dismantling, which does not always restore [6,8]. Such cleavage spindle disorders bring about aneuploidy, embryo development disturbances, and their implantation potential decrease. Embryos on cleavage stage are also very sensitive to temperature variations, they become tolerant to that only after compactization stage.

Short-time cultivation disorders aroused by embryo visual assay necessity also may be bound to the illuminant. Embryos are not affected by the illuminant under in vivo conditions. During in vitro cultivation also embryo normally stays away from light. Illumination impact has been reported capable of affecting embryos directly (DNA damage, mitochondrial degeneration, reactive oxygen intermediate occurrence in cytoplasm) [9] as well as indirectly, through medium component photooxidation (e.g. riboflavin disintegrated into tryptophan retards embryo cleavage) [6]. It has been also proved that light impact on paraffin oil may launch a cascade of peroxide reactions resulting in transferring water-soluble contaminants into culture medium [10].

To minimize the named factor impact, the transfer from one medium into another is accompanied by embryo development assay enabling to avoid multiple culture condition disorders and also extending the time of keeping embryos outside optimal conditions. Thus topical remains the issue of finding balance between cultivating condition stability and practical need of getting embryo development information.

Objective of this study is to estimate the effect on embryo development and implantation potential performed by short-term 3-day embryo culture condition disorders inevitably occurring due to light microscopy applied for morphological parameter assay.

Material and methods

Ovary stimulation in IVF controlled cycles was performed either within down-regulation long protocol involving gonadotrophin-releasing hormone agonist or applying short protocol with gonadotrophin-releasing hormone antagonist. The induced folliculogenesis control was carried out by ultrasound investigation. A follicle was considered functionally mature when reaching 17–18 mm size and no less than 300 pmol/l estradiol content in serum per each follicle exceeding 15 mm in diameter. The day for final follicle maturation triggering was determined based on the dominant follicle medium diameter (at least 17 mm) and endometrium thickness (no less than 8 mm).

Follicle transvaginal puncture (TVP) was performed 35–36 h after triggering of ovulation. The fertilization was carried out within 40 h after triggering (chorionic gonadotropin 3000–10000 ME). The fertilization assay was performed in 16–20 h. Embryos were cultivated in groups not more than 5 embryos in a culture medium droplet under mineral oil in three-step cultivation. On the 1st and 3rd day of embryo development, culture medium change was performed according to the producer’s recommendations. The embryo estimation on the 3rd and 5th day was performed according to Gardner’s classification [11] with their simultaneous transfer into next stage cultivation medium. Embryos of 730 (58%) patients were quickest possible transferred into next stage cultivation medium under binocular control without detailed morphological embryo description, while in 523 (42%) patient’s embryo quality assay was performed under inverted NIKON Ti microscope with further protocoling, which prolonged the period of residence outside the incubator for 30 s (per every 5 estimated embryos).

In accordance with the objective results of IVF program for 1253 patients with female sterility at the age of 23–46 years were examined for number of embryos having achieved blastocyst stage and for pregnancy rate(PR) index per 5th day development embryo transfers cases (detected by gestational sac ultrasonic study on the 21st day after embryo transfer). IVF program productivity was also estimated for 3-day (491 patient) and 5-day embryo transfers (1253).

The obtained data were processed applying variation statistic techniques, presented in R 3.3.2 program employing Fischer exact test. Bonferroni correction was applied to take into account the multiple comparisons effect. Pursuant thereto the value critical level (р) was .01.

Results

Basing on analyzed data one can see that embryo transfer was usually performed on 5th development day (72%) (see Table 1) whereby no PR statistical difference can be observed both for 3rd and 5th development day embryo transfers (Figure 1).

Figure 1. Pregnancy rate for 3rd and 5th development day embryo transfers (ET). – Proprietary data.

Table 1. Pregnancy rate for 3rd and 5th development day embryo transfers (ET).

	3rd-day transfer	5th-day transfer
Number of transfers	491	1253
Pregnancies	187	498
POF, %	38%	40%

We can also notice PR decrease in case of 3rd development day embryo estimation for older reproductive age (see Table 2) (average 10% decrease compared to the group where embryo estimation was not performed), yet this difference is not statistically authentic (Figure 2).

Figure 2. Pregnancy rate in different patient age groups with or without 3rd-day embryo estimation procedure.

Table 2. Pregnancy rate in different age groups for 5th-day development embryo transfers considering 5th-day development embryo estimation parameter.

Age	5th day (no estimation)			5th day (with estimation)
	ET (n)	Pregnancy (n)	PR%	ET (n)	Pregnancy (n)	PR %	p value
Before 30 years	168	75	45%	147	66	45%	1
31–35 years	319	145	45%	204	78	38%	.1233
36–39 years	187	64	34%	114	41	36%	.8035
40–42 years	49	17	35%	42	10	24%	.3575
More than 43 years	7	1	14%	16	1	6%	.5257

The analysis of obtained blastocyst number showed statistically authentic difference between cases with and without performed embryo quality estimation in patient groups below 39 years (Table 3, Figure 3). The difference is even higher for the age group after 36. No statistically authentic difference in obtained blastocyst number could be detected for the patient group after 40, which is perhaps bound to the group small size (56 patients without embryo estimation and 58 – with the estimation procedure).

Figure 3. Blastocyst formation frequency in different patient age groups with or without 3rd-day development embryo estimation procedure. [

– blastocyst number difference for the group depending on whether the estimation procedure was performed or not; statistically veracious (р<.01)].

Table 3. Blastocyst formation frequency in different patient age groups with or without 3rd-day development embryo estimation procedure.

Age	Without estimation			With estimation
	Fertilization (n)	Blastocyst (n)	Blastocyst, %	Fertilization (n)	Blastocyst (n)	Blastocyst %	p value
Before 30 years	1136	736	65%	457	233	51%	.000000420
31–35 years	2350	1379	59%	853	434	51%	.000090880
36–39 years	1104	674	61%	440	201	46%	.000000044
40–42 years	273	156	57%	88	45	51%	.3270000
More than 43 years	33	15	45%	23	6	26%	.1696000

Discussion

The pre-implantant period of embryo development appears to be one of the most sensitive periods for organism formation. In vitro cultivation is the most significant factor for epigenetiс reprograming and embryo development within IVF programs. A growing number of studies confirm the culture medium influence on the epigenetic preimplantant development. One of the supposed mechanisms are the possible methylation mistakes, occuring at 6–8 cell stage of embryo development [12–14].

There is a distinct difference in embryo stress reaction. The oocyte and cleavage stages are much more vulnerable than those after compactization [6] (Figure 4).

Figure 4. Embryo sensitivity to environmental factors on different formation stages [6].

Herewith it is hardly possible to restrain completely from any invasion taking into account the fertilization assay necessity.

According to the literature, as well as, to the present study data prolonged embryo culture before 5 days of development has no adverse impact on implantation potential compared to embryo culture and transfer on 3rd development day. The majority of scholars consider IVF productivity to be much higher when embryo transfer is performed on the 5th day which also witnesses the absence of any adverse impact of prolonged embryo cultivation.

However, our data show that when 3rd day embryo location period outside ‘ideal’ conditions increases it causes embryo blastulation percentage decrease and also affects the PR especially in elder patients group (however we did not succeed in obtaining statistically authentic data due to patients low number). The mentioned results are confirmed by the investigations showing embryo growing tolerance to culture condition changes after the compactization stage.

There are several ways of minimizing the adversary impact on embryos: switching to monostage culture mediums and also applying time-lapse microscopy systems. Despite all the advantages of monostage culture medium application it is pertinent to mention that first, they do not imitate embryo medium changes on its way within uterine tubes in vivo, and second, – continuous cultivation until 5th day of development leads to gradual accumulation of protein decay products under 37°С, including ammonium [15–17], which causes expression changes of a great number of genes involved in cell metabolism.

No doubt, three-stage culture medium application appears to be the optimal variant, however, it is worth restraining from additional embryo estimation on the 3rd development day in favor of minimizing ‘ideal’ condition disorders.

Conclusion

1. It has been established that pregnancy rate, as well as healthy child birth frequency, is higher in case of 5th-day development embryo transfer which speaks in favor of prolonged cultivation. Along with that, it is better to minimize the external factor impact on embryos. The 3rd-day embryo quality estimation is accompanied by keeping the embryo outside incubator which causes pH, temperature, and illuminant changes.

2. The 3rd-day embryo quality estimation does not influence much the PR and implantation potential. However, prolonged embryo location outside incubators along with their additional development quality estimation on the 3rd day definitely affects the number of obtained blastocysts, i.e. has an adversary impact on blastocyst formation especially in patients of older reproduction age.

Acknowledgments

The authors thank all the team members of the Moscow Region Research Science Obstetrics and Gynecology Institute, clinics ‘MK Semya’ and ‘Prior Clinic’ for providing assistance.

Disclosure statement

No potential conflict of interest was reported by the authors.