ORIGINAL ARTICLE


https://doi.org/10.5005/jogyp-11012-0016
Journal of Obstetric and Gynaecological Practices POGS
Volume 1 | Issue 2 | Year 2023

Comparison of Effects of Stimulation Days on Oocyte Morphology and Day 5 Blastocyst Rate in ICSI Cycle


Jayashree Babu1, Chandan Nagaraj2, Mahalakshmi Saravanan3, Nidhi Sharma4

1Department of Obstetrics and Gynaecology and Reproductive Medicine, Saveetha Medical College, Chennai, Tamil Nadu, India

2,3Department of Embryology, ARC International Fertility and Research Centre, Chennai, Tamil Nadu, India

4Department of Obstetrics and Gynaecology, Saveetha Medical College, Chennai, Tamil Nadu, India

Corresponding Author: Jayashree Babu, Department of Obstetrics and Gynaecology and Reproductive Medicine, Saveetha Medical College, Chennai, Tamil Nadu, India, Phone: +91 9003156986, e-mail: bjayashreer@gmail.com

How to cite this article: Babu J, Nagaraj C, Saravanan M, et al. Comparison of Effects of Stimulation Days on Oocyte Morphology and Day 5 Blastocyst Rate in ICSI Cycle. J Obstet Gynaecol Pract POGS 2023;1(2):49–53.

Source of support: Nil

Conflict of interest: None

Received on: 11 June 2023; Accepted on: 28 August 2023; Published on: 22 November 2023

ABSTRACT

Aim: To compare and identify the effect of normal and prolonged stimulation on oocyte morphology and day 5 blastocyst rate in intracytoplasmic sperm injection (ICSI) cycle in normal reserve patients.

Materials and methodology: A comparative study where two groups were considered—patients undergoing controlled ovarian stimulation for ICSI cycle with anti-müllerian hormone (AMH) 2–4 ng/mL with stimulation days of 10 and 11, and patients undergoing controlled ovarian stimulation for ICSI cycle with AMH 2–4 ng/mL with stimulation days of 12, 13, and 14 between March 2023 and May 2023. The mean AMH value was 2.91 ng/mL. The mean age of the patients in normal stimulation was 31.25 years and in prolonged stimulation was 33.11 years. Around 34 patients (403 oocytes) were analyzed in normal stimulation and 32 patients (303 oocytes) were analyzed in prolonged stimulation. Type of trigger, gonadotropin used, and the dosage were not considered.

Result: The oocyte maturation rate, fertilization rate, cleavage rate, day 3 good embryo rate, and day 5 blastocyst rate of normal stimulation are 76, 82, 76, 61, and 39%, respectively. The oocytes with single, double, and multiple defects of normal stimulation are 23, 55, and 22%, respectively. The oocyte maturation rate, fertilization rate, cleavage rate, day 3 good embryo rate, and day 5 blastocyst rate of prolonged stimulation are 76, 84, 76, 64, and 30%, respectively. The oocytes with single, double, and multiple defects of normal stimulation are 6, 41, and 77%, respectively.

The p-value for the oocyte maturation rate was 0.032535731 and the p-value for day 5 blastocyst rate was 0.003925882.

Conclusion: Prolonged stimulation is associated with oocyte maturation rate, oocyte morphology, and day 5 blastocyst rate.

Keywords: Anti-müllerian hormone, Controlled ovarian stimulation, Gonadotropin, Oocyte maturation.

INTRODUCTION

Couple who could not conceive after 12 months of unprotected and regular sexual intercourse is termed as infertile. This can be due to male or female factor or combined. Infertility can be primary or secondary. Primary infertility—if a couple has never conceived or they never had a baby before. Secondary infertility—couple’s inability to conceive despite having at least one child.1

Assisted reproductive technology (ART) is an artificial treatment that handles both eggs and sperm that facilitate pregnancy for the couples who are trying to conceive. Assisted reproductive techniques such as intrauterine insemination and in vitro fertilization (IVF) with or without intracytoplasmic sperm injection (ICSI) are the most successful therapeutic means for male factor infertility.2 The effective stimulation protocols and advanced culture techniques yield better grade embryos.3

Maturation of gametes occurs during puberty as shown in Figure 1. Sperm is formed by the process known as spermatogenesis and oocytes are formed by the process known as oogenesis.4

Fig. 1: Spermatogenesis and oogenesis

Sperm is produced by male reproductive system. Spermatogenesis takes place in the seminiferous tubules. The process involves proliferation of spermatogonia; spermatogonial differentiation into spermatocytes; meiotic division of spermatocytes producing spermatids; maturation of round spermatids; and the release of mature spermatozoa into the testicular tubule lumen. The entire process requires 42–76 days.5 Its structure and function is specialized in such a way that it reaches the oocyte during fertilization in vivo and in vitro.

Oocytes are produced by the female reproductive system. Oocytes are one of the largest cells in the human body. The intricate process of oogenesis is controlled by a myriad of intra- and extra-ovarian variables. Oogonia, which develop from primordial germ cells, multiply by mitosis to become primary oocytes. Until they are completely developed, these oocytes arrest at the prophase stage of the first meiotic division. The synthesis and accumulation of RNAs and proteins during oogenesis in primordial oocytes is vital for oocyte development and maturation as well as for the embryo’s ability to develop into a viable organism following fertilization. During folliculogenesis, oocyte meiotic and developmental competence is acquired gradually and sequentially.6

In the past, the term “ovarian reserve” has been used to refer to a woman’s capacity for reproduction, more precisely the quantity and quality of oocytes she possesses. Currently, the term refers to the number of oocytes that are still present rather than oocyte quality. The basal tests follicle-stimulating hormone (FSH), antral follicle count, and anti-müllerian hormone (AMH) are the markers that are most often utilized in clinical practice.7

Granulosa cells of small, developing follicles in the ovary release AMH. Anti-müllerian hormone has gained more attention as a measure for ovarian reserve as serum AMH levels substantially correspond with the number of developing follicles.8 Anti-müllerian hormone level less than 1 ng/mL is considered as low ovarian reserve.

Natural fertilization process involves sperm–oocyte fusion, sperm capacitation, acrosome reaction, sperm penetration into zona pellucida, and sperm penetration into perivitelline space. During ICSI, events such as zona reaction and sperm oocyte penetration are bypassed.9

In ICSI single morphologically normal sperm is selected and injected into the ooplasm of mature oocytes. Intracytoplasmic sperm injection is preferred for patients with low sperm count or for azoospermic patients where sperm are surgically retrieved. It yields high fertilization rates and pregnancy rates.

Controlled ovarian stimulation protocol ensures multifollicular development and retrieval of mature oocytes for IVF or ICSI. This increases the probability of fertility rate, blastocyst rate, and the success rate. Stimulation protocol is determined based on patient age, ovarian reserve, and ovarian response to the drugs administered. Protocols can be natural cycle, agonist cycle (long protocol), and antagonist cycle (short protocol).10

In antagonist protocol, the gonadotropin receptors are blocked in a dose-dependent competitive manner. This leads to suppression of gonadotropin secretion.10

Gonadotropins used while stimulating the patient can cause negative impacts on oocyte morphology.11 As the abnormal oocytes are matured during stimulation (which would have become atretic during the natural cycle), the oocyte quality gets affected.12

The result of studies regarding the effect of oocyte morphology abnormalities on fertilization rate, cleavage rate, and blastocyst rate remains controversial. Several studies show that transferring day 5 blastocysts results in greater pregnancy rates than transferring day 6 blastocysts, indicating faster-developing embryos have better viability. The aim of this study is to compare the number of days of stimulation and its effect on oocyte morphology and day 5 blastocyst rate in normal reserve patients (AMH: 2–4 ng/mL).

MATERIALS AND METHODOLOGY

All ICSI cycles at ARC International Fertility and Research Centre from March 2023 to May 2023 with AMH values between 2 and 4 ng/mL were involved. Inclusion criteria were age between 21 and 40 years, AMH 2–4 ng/mL, antagonist protocol, intra- and extracytoplasmic oocyte abnormalities. Exclusion criteria were polycystic ovarian syndrome (PCOS), low ovarian reserve, surgically retrieved sperms, donor sperm, donor oocyte, agonist protocol, E2 and P4 values, type of trigger, gonadotropin used, and gonadotropin dosage.

Two groups were considered—patients undergoing controlled ovarian stimulation for ICSI cycle with AMH 2–4 ng/mL with stimulation days of 10 and 11 (n = 34), and patients undergoing controlled ovarian stimulation for ICSI cycle with AMH 2–4 ng/mL with stimulation days of 12, 13, and 14 (n = 32). The mean AMH value was 2.91 ng/mL. The mean age of the patients in normal stimulation was 31.25 years and in prolonged stimulation was 33.11 years. Around 34 patients (403 oocytes) were analyzed in normal stimulation and 32 patients (303 oocytes) were analyzed in prolonged stimulation.

This study has been reviewed and approved by the Institutional Ethical Committee of Saveetha University-SMC/IEC/2021/12/007. All the patients were provided with written informed consent to use their gametes for this study.

Anti-müllerian Hormone Test

The blood sample was collected from the patient. The sample was then transferred into a tube that contains an anticoagulant to prevent clotting. The blood sample was processed to separate the serum or plasma, which is the liquid component of the blood that contains the hormones. This was usually done by centrifugation, which separates the blood cells from the liquid portion.

The VIDAS instrument was prepared for the test. This involves ensuring that the necessary reagents and consumables are available and properly loaded onto the instrument. The VIDAS system uses specific reagents and calibrators for the AMH assay. The processed serum or plasma sample was loaded onto the VIDAS instrument. The instrument has a loading mechanism that allows the operator to input the sample into the system. The VIDAS instrument performs the immunoassay. It uses antibodies that specifically bind to AMH in the sample. The antibodies were labeled with a fluorescent marker. During the test, the instrument measures the amount of fluorescence emitted, which is proportional to the concentration of AMH in the sample. The VIDAS system analyzes the fluorescence data obtained from the test and calculates the AMH concentration in the sample. The results were typically displayed on the instrument’s screen or printed out.

Controlled Ovarian Stimulation

Around 66 patients underwent antagonist (step down) protocol with AMH 2–4 ng/mL. Patients were stimulated with urinary FSH, folliculin—urofollitropin which has the activity of FSH (Bharath Serums and Vaccines Limited, Ambernath) or recombinant FSH, Follisurge (Intas Pharmaceuticals Limited, Ahmedabad) or urinary HMG, IVF M (Cipla, Mumbai). Antagonist such as Ciscure (Cetrorelix Acetate, Emcure Pharmaceuticals Limited, Hinjawadi, Pune) or Ovurelix (Cetrorelix Acetate, Sun Pharmaceutical Industries Limited, Mumbai) were administered when the follicle reaches 12–14 mm, around day 5 of stimulation. When the follicle reaches 18–20 mm, trigger was given. Based on follicle count and E2 level, type of trigger was decided. Patients with high E2 level (>6000 pg/mL) and follicle count were administered with agonist trigger, decapeptyl 0.2 mg (Triptorelin Acetate, Ferring Pharmaceuticals Limited, Thane). Patients with E2 <5000 pg/mL and low follicle count, were administered with Ovitrelle 250 µg (Recombinant HCG, Merck, Bhiwande). Patients with low follicle count and high E2 level were administered with dual trigger (HCG 5000 IU and leuprolide 0.2 mg).

Sample Preparation

Semen analysis was done according to the WHO 6th edition manual. Samples were obtained by ejaculation. Cell counter and light microscope were used to determine sperm count and motility. Morphology was evaluated by Diff Quik staining. All the samples were prepared by density gradient, simple wash, swim up, or cell sorter. The prepared sample was incubated at 37ºC in 6% CO2 until use.

Oocyte Retrieval, Denudation and ICSI

Follicular fluid was retrieved using single lumen oocyte retrieval (OCR) needle (Fig. 2). Follicular fluid was screened and cumulus oocyte complex (COC) were collected and incubated for about 2 hours before denudation. Oocytes were denuded with 80 IU/mL hyaluronidase (Vitromed, Langenfeld, Germany) along with mechanical stripping using denuding pipettes. After denudation, M II oocytes were washed with HEPES (Vitromed, Langenfeld, Germany). Intracytoplasmic sperm injection was carried out on the heated stage (37°C) of an inverted microscope (Olympus, Japan) at 400X magnification using Narishige micromanipulator (Sony Corporation, Japan; Fig. 3). Morphologically normal sperms will be selected for ICSI.

Fig. 2: Ovum pickup needle (single lumen)

Fig. 3: Intracytoplasmic sperm injection micromanipulator

Oocyte Evaluation

Morphology of the oocytes was assessed just before ICSI by inverted microscope. Oocytes with single defect, double defect, and multiple defects were noted.

Assessment of Fertilization, Embryo Cleavage and Blastocyst Formation

The injected oocytes were incubated in culture media (One step, Vitromed, Langenfeld, Germany). Around 16–18 hours post ICSI, fertilization was evaluated by determining the presence of 2 polar body (PB) or 2 pronucleus (PN). About 66–69 hours post ICSI (day 3) embryos were evaluated based on number of blastomeres and degree of fragmentation. After 106–108 hours post-ICSI (day 5), blastocyst quality was assessed by Gardener grading.

Number of M II oocytes retrieved, no fertilized, cleaved, day 3 good embryos and day 5 blastocyst from both the groups were noted.

Statistical Analysis

The statistical analysis was done to compare the oocyte maturation rate and day 5 blastocyst rate in normal stimulation group and prolonged stimulation group. The numerical data were analyzed using two sample t-test with unequal variances. P-values of < 0.05 were considered significant. MS Excel was used to analyze the data.

The oocyte morphology defects, fertilization rate, cleavage rate, and day 3 good embryos rate were represented in bar graph.

RESULTS

Normal stimulation had 34 patients with 403 oocytes retrieved out of which 305 oocytes were M II, 250 fertilized, 233 cleaved, 186 day 3 good embryos, 118 day 5 blast. Prolonged stimulation had 32 patients with 303 oocytes retrieved out of which 230 oocytes were M II, 194 fertilized, 174 cleaved, 147 day 3 good embryos, 69 day 5 blast (Fig. 4). In normal stimulation, 70 oocytes had single defects, 169 oocytes had double defects, and 66 oocytes had multiple defects, and in prolonged stimulation 13 oocytes had single defects, 41 oocytes had double defects, and 176 oocytes had multiple defects (Fig. 5).

Fig. 4: Effects of normal stimulation and prolonged stimulation in fert rate, cleave rate, day 3 good embryo rate, and day 5 blastocyst rate

Fig. 5: Effect of normal stimulation and prolonged stimulation on oocyte morphology

The oocyte maturation rate, fertilization rate, cleavage rate, day 3 good embryo rate, and day 5 blastocyst rate of normal stimulation is 76, 82, 76, 61, and 39%, respectively. The oocytes with single, double, and multiple defects of normal stimulation are 23, 55, and 22%, respectively. The oocyte maturation rate, fertilization rate, cleavage rate, day 3 good embryo rate, and day 5 blastocyst rate of prolonged stimulation is 76, 84, 76, 64, and 30%, respectively. The oocytes with single, double, and multiple defects of normal stimulation are 6, 41, and 77%, respectively.

The p-value for the oocyte maturation rate was 0.032535731 which is less than 0.05. Hence there is a significant difference between the effect of normal stimulation and prolonged stimulation on oocyte maturation (Table 1).

Table 1: Analysis using MS Excel for oocyte maturation
t-test: Two-sample assuming unequal variances
  Variable 1 Variable 2
Mean 9.53125 6.764705882
Variance 29.93447581 22.60962567
Observations 32 34
Hypothesized mean difference 0  
df 62  
t-stat 2.186843566  
p (Tt) one-tail 0.01626786  
T critical one-tail 1.669804163  
p (Tt) two-tail 0.032535731  
t critical two-tail 1.998971517  

The p-value for day 5 blastocyst rate was 0.003925882 which is less than 0.05. Hence there is a significant difference between the effect of normal stimulation and prolonged stimulation day 5 blastocyst rate (Table 2).

Table 2: Analysis using MS Excel for day 5 blastocyst rate
t-test: Two-sample assuming unequal variances
  Variable 1 Variable 2
Mean 3.6875 2.02941176
Variance 7.899193548 1.6657754
Observations 32 34
Hypothesized mean difference 0  
df 43  
t-stat 3.048434357  
p (Tt) one-tail 0.001962941  
T critical one-tail 1.681070703  
p (Tt) two-tail 0.003925882  
t critical two-tail 2.016692199  

DISCUSSION AND CONCLUSION

Ovarian stimulation is a critical step in ART procedures and requires close monitoring, individualized dosing, and experienced medical guidance. Fertility specialists work to balance the goals of achieving an adequate number of mature eggs while minimizing the risk of complications. Ovarian stimulation aims to increase the number of available eggs for retrieval, increasing the chances of successful fertilization, and embryo development. The longer stimulation protocol affects the quality of embryos, leading to a higher proportion of lower-grade or lower-quality blastocysts. This can impact the success rates of implantation and pregnancy.

For successful fertilization, subsequent embryo development, and pregnancy to occur, oocyte maturity is crucial. For this luteinizing hormone (LH) and FSH must act sufficiently on both theca cells and granulosa cells. Oocyte maturation rate was often calculated by dividing the number of M II oocytes by the total number of oocytes. Only nuclear maturation, or the resumption and progression of meiosis to M II, was represented by it.13

The euploid rate of day 5 blastocysts is significantly higher than that of day 6 blastocysts.14 Day 5 blastocysts in oocyte donation program have significantly higher clinical pregnancy rates (CPRs) and live birth rates (LBRs), and present shorter time to delivery, compared to day 6 blastocysts, regardless of embryo quality.15

In our study, the oocyte maturation rate, oocyte morphology, fertilization rate, cleavage rate, day 3 good embryo rates, and day 5 blastocyst rate were affected in prolonged stimulation group. Therefore we suggest that the duration of stimulation affects the oocyte maturation rate, oocyte morphology, and day 5 blastocyst rate.

The limitation of this study is low sample size. The lead follicle size of the patients was not taken into consideration. The type of gonadotropin (urinary or recombinant) and dose of the gonadotropin were not included. Further studies have to be done to confirm the result.

REFERENCES

1. World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edition. Geneva: World Health Organization; 2010.

2. Schiewe MC An effective, simplified, and practical approach to intracytoplasmic sperm injection at multiple IVF centers. J Assist Reprod Genet 13(3):238–245. DOI: 10.1007/BF02065943.

3. Fancsovits P, Tóthné ZG, Murber Á, et al. Correlation between first polar body morphology and further embryo development. Biologia Futura 2006; 57(3):331–338.DOI: 10.1556/ABiol.57.2006.3.7.

4. Finlayson C, Johnson E, Chen D et al. Proceedings of the Working Group Session on fertility preservation for individuals with gender and sex diversity. Transgender Health 2016;1(1):99–107. DOI: 10.1089/trgh.2016.0008.

5. Neto FT, Bach PV, Najari BB, et al. Spermatogenesis in humans and its affecting factors. Semin Cell Dev Biol 2016;59:10–26. DOI: 10.1016/j.semcdb.2016.04.009.

6. Sánchez F, Smitz J. Molecular control of oogenesis. Biochim Biophys Acta 2012;1822(12):1896–1912. DOI: 10.1016/j.bbadis.2012.05.013.

7. Tal R, Seifer DB. Ovarian reserve testing: A user’s guide. Am J Obstet Gynecol 2017;217(2):129–140. DOI: 10.1016/j.ajog.2017.02.027.

8. Moolhuijsen LME, Visser JA. Anti-müllerian hormone and ovarian reserve: Update on assessing ovarian function. J Clin Endocrinol Metab 2020;105(11):3361–3373. DOI: 10.1210/clinem/dgaa513.

9. Rao KA. Principle and Practice of Assisted Reproductive Technology, 1st edition. New Delhi: Jaypee Brothers Medical Publication; 2014.

10. Talwar P. Jaypee’s Video Atlas of Assisted Reproductive Technologies and Clinical Embryology, 1st edition. New Delhi: Jaypee Brothers Medical Publication; 2014. p. 406.

11. Taheri F, Alemzadeh Mehrizi A, Khalili MA, et al. The influence of ovarian hyperstimulation drugs on morphometry and morphology of human oocytes in ICSI program. Taiwan J Obstet Gynecol 2018;57(2):205–210. DOI: 10.1016/j.tjog.2018.02.007.

12. Ebner T, Yaman C, Moser M, et al. Prognostic value of first polar body morphology on fertilization rate and embryo quality in intracytoplasmic sperm injection. Human Reprod 2000;15(2):427–430. DOI: 10.1093/humrep/15.2.427.

13. Yu-Chieh Yang, Yi-Ping Li, Song-Po Pan, et al. The different impact of stimulation duration on oocyte maturation and pregnancy outcome in fresh cycles with GnRH antagonist protocol in poor responders and normal responders. Taiwan J Obstet Gynecol 58(4):471–476. DOI: 10.1016/j.tjog.2019.05.007.

14. Tong J, Niu Y, Wan A, et al. Comparison of day 5 blastocyst with day 6 blastocyst: Evidence from NGS-based PGT-A results. J Assist Reprod Genet 2022;39(2):369–377. DOI: 10.1007/s10815-022-02397-0.

15. Yerushalmi GM, Shavit T, Avraham S, et al. Day 5 vitrified blastocyst transfer versus day 6 vitrified blastocyst transfer in oocyte donation program. Sci Rep 2021;11(1):10715. DOI: 10.1038/s41598-021-90238-y.

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