ORIGINAL ARTICLE


https://doi.org/10.5005/jogyp-11012-0014
Journal of Obstetric and Gynaecological Practices POGS
Volume 1 | Issue 2 | Year 2023

Impact of Oocytes with Mild and Heavy Debris in Perivitelline Space on Blastocyst Quality


Dinesh Ram V1https://orcid.org/0009-0005-5196-9529, Chandan N2, Mahalakshmi3, Jayashree Srinivasan4, Damodharan J5

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

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

4,5Department of Reproductive Medicine, Saveetha Medical College, Chennai, Tamil Nadu, India

Corresponding Author: Dinesh Ram V, Department of Obstetrics and Gynaecology, Saveetha Medical College, Chennai, Tamil Nadu, India, Phone: +91 6381446552, e-mail: vldineshram2812@gmail.com

How to cite this article: Ram VD, Chandan N, Mahalakshmi, et al. Impact of Oocytes with Mild and Heavy Debris in Perivitelline Space on Blastocyst Quality. J Obstet Gynaecol Pract POGS 2023;1(2):42–48.

Source of support: Nil

Conflict of interest: None

Received on: 29 July 2023; Accepted on: 30 August 2023; Published on: 22 November 2023

ABSTRACT

Aim: The aim of this study is to compare the blastocyst quality of oocytes with mild and heavy debris in perivitelline space (DPVS).

Objective: To investigate the rate of blastocyst development of oocytes with mild DPVS and to investigate the rate of blastocyst development of oocytes with heavy DPVS, then to compare the rate of blastocyst development of oocytes with mild and heavy DPVS and to identify ideal blastocyst for transfer.

Study population: Patients who have undergone controlled ovarian stimulation for the intracytoplasmic sperm injection (ICSI) cycle from March 2023 to May 2023 ARC International Fertility and Research Centre.

Study groups: Two groups were considered—oocytes with mild debris or granularity in perivitelline space (PVS) and oocytes with heavy debris or granularity in PVS.

Results: This study from March 2023 to May 2023 was done at ARC International Fertility and Research Centre. Patients undergoing the Antagonist protocol for the ICSI cycle were considered. Around 371 M2 oocytes were analyzed, in that 203 had mild debris or granularity in PVS and 168 had heavy debris or granularity in PVS.

In oocytes with mild debris or granularity in PVS, their fertilization rate, cleavage rate, and frozen blastocyst rate are 88, 81, and 47%, respectively. In oocytes with heavy debris or granularity in PVS their fertilization rate, cleavage rate, and frozen blastocyst rate are 76, 59, and 15%, respectively.

Oocytes with heavy debris or granularity in PVS have compromised blastocyst quality compared with mild debris or granularity in PVS.

Conclusion: In this study, we conclude that debris or granularity in PVS can be considered an indicator of oocyte competence. This can help in identifying a cohort of oocytes with a lower chance of forming viable embryos. This helps in reducing the culture of supernumerary embryos. We suggest that when the patients enroll for donor oocytes, oocytes without heavy debris or granularity in PVS should be used because heavy granularity in PVS has compromised blastocyst conversion. Further studies are needed with a large sample size to get a conclusion.

Keywords: Human menopausal gonadotropin, Perivitelline space, Zona pellucida.

INTRODUCTION

Fertility in humans refers to the ability to conceive and reproduce. It involves several factors, including the quality and quantity of gametes (sperm and eggs), the reproductive health of both partners and the timing of intercourse.14

Infertility is a common medical problem, where the couple cannot conceive within one year of active sexual intercourse. The fertility investigations for females include evaluation of ovulation and tubal patency investigations for males include semen parameters. Infertility can be due to male factor or female factor or combined factors (WHO 1999).58 In developed countries, around 2–4% of births occur due to assisted reproductive technology (ART).

Intrauterine insemination (IUI), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI) come under ART which has therapeutic means for infertility.912

The male reproductive system functions to produce (spermatogenesis) and deposit sperm.13 The sperm carries 23 pairs of chromosomes and a centrosome. The female reproductive system functions to produce oocytes (oogenesis) and to protect and nourish the fetus until birth. The oocyte is one of the largest cells in the human body.14,15

Human reproduction involves the fertilization of sperm and egg which occurs in the ampulla of the fallopian tube.1619 This results in the production of a zygote or a fertilized oocyte. The stages of fertilization can be divided into sperm oocyte fusion, sperm capacitation, acrosome reaction, sperm penetration into zona pellucida (ZP), and sperm penetration into PVS.20

During ICSI the sperm is directly injected into the ooplasm, which leads to bypassing the zona reaction and sperm oocyte penetration.2125 Furthermore, ICSI increases the probability of fertilization rate which leads to an increase in success rate.26

Sperm have three main structural regions: the head, midpiece, and tail (flagellum). While the midpiece and tail provide the motility necessary for the spermatozoon to reach the site of fertilization, the sperm head contributes its haploid set of chromosomes to the oocyte at fertilization. The sperm head contains the nuclear DNA which is heavily condensed by the associations between DNA and protamines.2732

Controlled ovarian stimulation protocol ensures retrieval of mature oocytes for IVF or ICSI. Protocols such as agonist (long protocol) and antagonist (short protocol) are commonly followed. Agonist protocol involves suppression of the hypothalamic–pituitary–ovarian (HPO) axis. Antagonist protocol involves blockage of gonadotropin-releasing hormone (GnRH) receptors which inhibits gonadotropin secretion. The trigger is given when the follicle reaches 17–18 mm. The trigger given can be either human choriogonadotropin (HCG) or an agonist trigger. Human choriogonadotropin has an alpha subunit and a beta subunit.3335 The alpha subunit is similar to that of LH and hence binds to LH receptors. Agonist trigger helps in reducing the risk of ovarian hyperstimulation syndrome (OHSS) but may cause luteal phase defect. The trigger is given to loosen the cumulus cells from the follicular wall; this facilitates the release of oocytes after 34–36 hours.3638

Oocyte is a cell in an ovary that may undergo meiotic division to form an ovum. Its size ranges from 100 to 120 µm. Oocyte with normal morphology has expanded cumulus with radiant corona, bright cytoplasm, intact polar body (PB), ZP with thickness 18–22 µm, perivitelline space (PVS) with no granularity.3945

Oocyte dysmorphism can be divided into intracytoplasmic anomalies, extracytoplasmic anomalies, and oocytes with abnormal size and shape.

Oocyte morphology can be assessed to find viable embryos. Embryo grading results may vary between clinics, and grading of day 2 or day 3 embryos or blastocysts remains challenging. Assessing oocyte morphology reduces the culture time (helps in the selection and culturing of viable embryos) and zona hardening can be avoided. This helps in identifying the ideal blastocyst for transfer. Identifying ideal blastocyst leads to better pregnancy rates.

What is Perivitelline Space?

Perivitelline space is the space between ZP and the cell membrane of an oocyte in which the extruded first PB is present. Cortical granules released from the ovum get deposited in PVS to block polyspermy (in normal fertilization). If the PVS is normal, the ooplasm is slightly away from the ZP in the surrounding area of the PB.

Abnormalities in PVS can be large PVS (in particular side or around the oocyte), granular or debris in perivitelline space (DPVS), or absence of PVS. Factors such as female age did not affect PVS, but the ratio of estradiol to testosterone (and to progesterone) did. Granularity in PVS is related to maturation events. Some of the studies proved the correlation between doses of human menopausal gonadotropin (HMG) and granularity in PVS. In some cases, the remnants of coronal cells deposit as granules in PVS, which usually withdraws when meiosis resumes. Studies regarding the correlation between granularity in PVS and embryo quality remain controversial. Various authors studied the correlation between granularity in PVS and fertilization rate, blastocyst quality, and pregnancy rate. The aim of this study is to assess the blastocyst quality of oocytes with mild and heavy DPVS.

AIM

The aim of this study is to compare the blastocyst quality of oocytes with mild and heavy DPVS.

OBJECTIVE

To enhance the pregnancy rate.

MATERIALS AND METHODOLOGY

Study Design

Prospective analytical study.

Study Setting

ARC International Fertility and Research Centre.

Inclusion Criteria

Age, 21–45 years; BMI, 18–38 kg/m2; marital life, above 5 years; previous IUI failure; sterilization/unexplained infertility; tubal anomaly and polycystic ovarian disease (PCOD); antagonist protocol; extracytoplasmic abnormalities (DPVS or granularity in PVS); oocyte with normal morphology; and sperm quality mild/moderate (oligo-, astheno-, and teratozoospermia); donor sperm.

Exclusion Criteria

Vitrified/thawed oocytes; surgical sperm retrieval; agonist protocol; and other oocyte abnormalities.

Study Population

Patients who have undergone Controlled ovarian stimulation for the ICSI cycle from March 2023 to May 2023 International Fertility and Research Centre.

This study has been reviewed and approved by the institutional ethical committee of Saveetha University (SMC/IEC/2021/12/007). After ensuring that the patients satisfied the inclusion and exclusion criteria, all the patients were provided with written informed consent to use their gametes for this study.

Study Groups

Two groups were considered—oocytes with mild debris or granularity in PVS and oocytes with heavy debris or granularity in PVS.

Controlled Ovarian Hyperstimulation

Stimulation was started from day 2 of the menstrual cycle. The patients will undergo the antagonist protocol, in which they will receive stimulation using different medications. These medications include urinary follicle-stimulating hormone (FSH), folliculin–urofollitropin (Bharath Serums and Vaccines Limited, Ambernath, Maharashtra, India), recombinant FSH, follisurge (Intas Pharmaceuticals Limited, Ahmedabad, Gujarat, India), or urinary HMG, IVF M (Cipla, Mumbai, Maharashtra, India). At around day 5 of stimulation, when the follicle reaches 12–14 mm, an antagonist such as Ciscure (Cetrorelix acetate, Emcure Pharmaceuticals Limited, Hinjewadi, Pune, Maharashtra, India) or Ovurelix (Cetrorelix acetate, Sun Pharmaceutical industries limited, Mumbai, Maharashtra, India) will be administered. Once the follicles reach 18–20 mm, a trigger will be given. The type of trigger administered will be decided based on the follicle count and E2 level. Patients with a high E2 level (>6000 pg/mL) and follicle count will receive an agonist trigger, Decapeptyl 0.2 mg (Triptorelin Acetate, Ferring Pharmaceuticals Limited, Thane). Patients with E2 < 5000 pg/mL and a low follicle count will receive Ovitrelle 250 µg (Recombinant HCG, Merck, Bhiwandi, Maharashtra, India). Patients with a low follicle count and high E2 level will receive a dual trigger (HCG 5000 IU and Leuprolide 0.2 mg).46

Sample Preparation

Semen Analysis was done based on the WHO Manual for Human Semen Analysis, 6th edition. Samples will be obtained by ejaculation. Cell counter and light microscope will be used to determine sperm count and motility (Fig. 1). Morphology will be evaluated by eosin staining. All the samples will be prepared either by density gradient or simple wash. The prepared sample will be incubated at 37°C in 6% CO2 until used it.

Fig 1: Sperm counting chamber

Oocyte Retrieval and Denudation

Follicular fluid will be screened and cumulus oocyte complex (COC) will be collected using sterile glass pipettes (Fig. 2) and incubated in culture media for about 2 hours before denudation. The cumulus complex was denuded with HYDASE (Vitromed, Langenfeld, Germany) using mechanical stripping using denuding pipettes. After denudation, M II oocytes will be washed with HEPES (Vitromed, Langenfeld, Germany) After washing, Oocyte morphology was assessed using a microscope. Oocytes with other morphological abnormalities were excluded.

Fig. 2: Sterile glass pipette for pickup

Intracytoplasmic Sperm Injection

Intracytoplasmic sperm injection will be carried out on the heated stage (37°C) of an inverted microscope (Olympus, Japan) at 400× magnification using a Narishige micromanipulator (Sony Corporation, Japan). Morphologically normal sperms will be selected for ICSI. Also, ICSI was done 2 hours postdenudation. Polyvinylpyrrolidone (PVP; Vitromed, Langenfeld, Germany) was used to decrease sperm motility. Holding and injection pipettes (Vitromed, Langenfeld, Germany) were used. Holding injection was used to hold the oocyte, injection needle was used to inject the sperm. After insemination, oocytes with mild and heavy debris were cultured in separate wells in a culture dish containing step media (one step; Vitromed, Langenfeld, Germany) droplet overlayer by oil (paraffin oil; Vitromed, Langenfeld, Germany).

Assessment of Fertilization: Embryo Cleavage and Blastocyst Formation

About 16–18 hours post-ICSI (day 1), fertilization check was done. Presence of two pronuclei (2PN) or two polar bodies (2PB) was considered normal fertilization. Presence of one polar body (1PN) or three pronuclei (3PN) was considered abnormal fertilization. Unfertilized oocytes and degenerate oocytes (damaged oocyte inside ZP) was also observed during fertilization check. Only the zygotes with 2PN or 2PB forms viable embryos.

Based on the number of cells, rate of fragmentation, symmetric blastomeres and absence of multi nucleation day 3 embryos will be assessed. About 68 ± 1-hours post-ICSI (day 3) embryo cleavage was assessed.

About 116 ± 2 hours post-ICSI (day 5) blastocysts develop. Blastocyst can be observed on day 5 and day 6. However, day-5 blastocysts are more viable than day 6 blastocysts. According to the Gardner grading system blastocyst was graded.

About 371 M2 oocytes were analyzed, and in that, 203 has mild debris or granularity in PVS (Fig. 3) and 168 has heavy debris or granularity in PVS (Fig. 4). Fertilization, cleavage, and blastocyst from both the groups were observed and noted separately.

Fig. 3: Mild granularity in PVS

Fig. 4: Heavy granularity in PVS

Statistical Analysis

The p-value defines the probability of getting a result that is either the same or more extreme than the other actual observations. The p-value represents the probability of occurrence of the given event. If the p-value is 0.05 or lower, the result is declared as significant, but if it is higher than 0.05, the result is non-significant.47,48

Two-sample t-test with unequal variance is a two-sample location test which is used to test the (null) hypothesis that two populations have equal means. This test is often referred to as “unpaired” or “independent samples” t-tests and is more reliable when the two samples have unequal variances and possibly unequal sample sizes.4954

Frequency refers to the number of times an event or a value occurs. A frequency table is a table that lists items and shows the number of times the items occur.5558

The statistical analysis was done to compare the blastocyst rate of oocytes with mild debris or granularity in PVS and heavy debris or granularity in PVS (Figs 5 to 8). The statistical analysis was done using two-sample t-test with unequal variances; p < 0.05 were considered significant.59

Fig. 5: Number of M2 oocytes with mild debris; number of oocytes fertilized, cleaved, and blastocysts

Fig. 6: Pie chart representing number of M2 oocytes with mild debris; number of oocytes fertilized, cleaved, and blastocysts

Fig. 7: Number of M2 oocytes with heavy debris

Fig. 8: Pie chart representing number of M2 oocytes with heavy debris; number of oocytes fertilized, cleaved, and blastocysts

The fertilization rate, cleavage rate, and day-3 good embryos rate were presented in a frequency table. Microsoft Excel was used for statistical analysis.60

Comparison of Blastocyst Rate of Oocytes with Mild Debris or Granularity in PVS and Heavy Debris or Granularity in PVS and Heavy DPVS

Hypothesis

  • Null hypothesis: There is no significant difference between the effect of mild and heavy debris.

  • Alternative hypothesis: Heavy debris has a negative impact on blastocyst rate.

Two-sample t-test with unequal variances (Table 1).

Table 1: Analysis using MS Excel; p-value: 0.00000896
  Variable 1 Variable 2
Mean 4 0.961538
Variance 6.173913 2.518462
Observations 24 26
Hypothesized mean difference 0  
df 39  
t-statistics 5.1060431  
p (Tt) one tail 0.00000448  
t-critical one tail 1.6848751  
p (Tt) two tails 0.00000896  
t-critical two tails 2.0226909  

Interpretation

The null hypothesis is rejected because the p-value (0.00000896) is smaller than the level of significance (0.05). Hence, the oocytes with heavy debris or granularity in PVS affected the blastocyst rate (Tables 2 to 7).

Table 2: Pivot table
Row labels Sum of fertilization Sum of cleavage Sum of blastocyst
Heavy 128 99 25
Mild 179 164 96
Grand total 307 263 121
Table 3: Fertilized rate, mean value
  Fertilized rate
Mild Heavy
Average 7.458333 4.923077
Median 7 3.5
Mode 6 1
Variance 14.51993 23.27385
Standard 3.810502 4.824297
deviation    
Mean value for mild DPVS: 7.458333; Mean value for heavy DPVS: 4.923077
Table 4: Cleavage rate, mean value
  Cleavage rate
Mild Heavy
Average 6.833333 3.807692308
Median 7 2
Mode 2 1
Variance 12.31884 18.88153846
Standard 3.509821 4.345289226
deviation    
Mean value for mild DPVS: 6.833333; Mean value for heavy DPVS:3.807692308
Table 5: Blastocysts rate, mean value
  Blastocysts rate
Mild Heavy
Average 6.833333333 3.807692
Median 7 2
Mode 2 1
Variance 12.31884058 18.88154
Standard deviation 3.509820591 4.345289
Mean value for DPVS: 6.833333333; Mean value for heavy DPVS: 3.807692
Table 6: Number of M2 oocytes with mild debris, number of oocytes fertilized, cleaved, and blastocysts
Observation
Mild debris or granularity in PVS group
Total number of M2 oocytes 203
Number of fertilized oocytes 179 (88%)
Number of cleaved oocytes 164 (81%)
Number of blastocysts 115 (57%)
Number of frozen blastocysts 96 (47%)
Table 7: Number of M2 oocytes with heavy debris; number of oocytes fertilized, cleaved, and blastocysts
Heavy DPVS or granularity in PVS group
Total number of M2 oocytes 168
Number of fertilized oocytes 128 (76%)
Number of cleaved oocytes 99 (59%)
Number of blastocysts 39 (23%)
Number of frozen blastocysts 25 (15%)

RESULTS

This study from March 2023 to May 2023 was done at ARC International Fertility and Research Centre. Patients undergoing Antagonist protocol for ICSI Cycle were considered. Around 371 M2 oocytes were analyzed, in that 203 has mild debris or granularity in PVS and 168 has heavy debris or granularity in PVS.

In oocytes with mild debris or granularity in PVS, their Fertilization rate, cleavage rate, and frozen blastocyst rate are 88, 81, and 47%, respectively. In oocytes with heavy debris or granularity in PVS, their fertilization rate, cleavage rate, and frozen blastocyst rate are 76, 59, and 15%, respectively.

Oocytes with Heavy debris or granularity in PVS has compromised blastocyst quality compared with mild debris or granularity in PVS.

DISCUSSION

Assessing the quality of oocytes helps in minimizing the culture time which benefits both patients and clinics.6163 Also preselecting the embryos based on oocyte morphology can be done on the day of OCR by non-invasive selection. Some of the countries such as Italy have regulations for ART, where only three oocytes can be inseminated per cycle. Insemination of all the oocytes and embryo cryopreservation is outlawed. Hence selection of good-quality oocytes for ICSI plays a significant role in the above-mentioned cases.64,65

From the results of certain studies, the effect of extra-cytoplasmic abnormalities remains controversial. Most of the studies had concluded that DPVS has compromised blastocyst quality but in some of the cohort studies blastocyst quality was moderate. Till now there is no study comparing mild and heavy debris.66,46

CONCLUSION

In this study, we conclude that debris or granularity in PVS can be considered as indicator of oocyte competence. This can help in identifying a cohort of oocytes with a lower chance of forming viable embryos. This helps in reducing the culture of supernumerary embryos.

We suggest that when the patients enroll for donor oocytes, oocytes without heavy debris or granularity in PVS should be used because heavy granularity in PVS has compromised blastocyst conversion. In this study, we analyzed around 371 oocytes; further studies need to be done with a large sample size to get a conclusion.67,68

ORCID

Dinesh Ram V https://orcid.org/0009-0005-5196-9529

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