Is embryogenesis and ART outcome different in polycystic ovary syndrome?

Madhuri Patil; Priyanka Reddy; Chinmayie Chandrashekar; Milind Patil

doi:10.4103/tofj.tofj_20_19

ORIGINAL ARTICLE

Year : 2019 | Volume : 2 | Issue : 2 | Page : 79-86

Is embryogenesis and ART outcome different in polycystic ovary syndrome?

Madhuri Patil, Priyanka Reddy, Chinmayie Chandrashekar, Milind Patil
Dr. Patil's Fertility and Endoscopy Clinic, Bengaluru, Karnataka, India

Date of Submission	05-Dec-2019
Date of Acceptance	12-Dec-2019
Date of Web Publication	31-Jan-2020

Correspondence Address:
Dr. Madhuri Patil
Dr. Patil's Fertility and Endoscopy Clinic, Bengaluru, Karnataka
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/tofj.tofj_20_19

Abstract

Introduction: As oogenesis is a continuum of months, oocyte quality will be dictated by events that occur during both the growth and maturation stages of development. Morphological variation of the oocyte and embryo result from intrinsic factors such as age and genetic defects or extrinsic factors such as stimulation protocol, culture condition, and nutrition. Controlled ovarian stimulation (COH) interferes with the balance of forces within the ovary and overrides their endogenous pattern of control to optimize normal selection process.
Aim: 1. To compare the embryogenesis and outcome of assisted reproductive technology (ART) in polycystic ovary syndrome (PCOS) and non-PCOS women. 2. To determine whether embryo quality, implantation rate (IR) and clinical pregnancy rate (CPR) are related to estradiol (E2) or progesterone (P4) levels on the day of human chorionic gonadotropin (hCG).
Materials and Methods: This was a retrospective case–control study of 425 women at a Tertiary Care Center from January 2017 to December 2018. Of these 183 were PCOS and 242 non-PCOS matched for body mass index and age. We compared the follicle stimulating hormone, antral follicle count (AFC), anti-Mullerian hormone, thyroid-stimulating hormone, total gonadotropin utilized, total days of stimulation, estradiol (E2), and progesterone (P4) on the day of hCG trigger between both groups. Before entering the in vitro fertilization program, patients were classified into PCOS and non-PCOS depending on the AFC at the baseline scan done on day 2/3 of the menstrual cycle. We calculated the fertilization, cleavage, blastocyst formation rate, and utilization rate of the embryos from the total oocytes retrieved and fertilized in four different groups classified according to the E2 levels on the day of the hCG (≤1000, 1001–2000, 2001–3000, >3000 pg/ml) in both PCOS and non-PCOS women. We also looked at the IR and CPR in the above four groups in fresh cycle and included the frozen embryo transfer cycles to calculate the utilization rate. Statistical comparison was done using the Mann–Whitney U test.
Results: There was no statistically significant difference in the fertilization rate (P = 0.803), cleavage rate was higher in PCOS group (P = 0.001) and blastocyst formation rate was higher in non-PCOS group (P < 0.001). There was no statistical difference seen in the grade of the embryos on day 3 and day 5 across the E2 groups in the PCOS women. The quality of embryos on D3 and D5 was much better in non-PCOS group with E2 levels more than 2000 pg/ml. The IR was similar in both PCOS and non-PCOS group when E2 values were <2000 pg/ml and >3000 pg/ml. The IR was higher in the PCOS group as compared to non-PCOS in women with E2 between 2000 and 3000 (P = 0.020). No difference in the CPR across the E2 groups. The utilization rate of the embryos per egg retrieved and per 2PN was statistically higher in patients with an E2 <1000 pg/ml in PCOS women. The utilization rate of the embryos per egg retrieved and per 2PN was significantly higher in non-PCOS when E2 was more than 2000 pg/ml.
Conclusion: As embryogenesis and endometrial receptivity can be affected by high E2 and P4 levels it is highly recommended to closely monitor COS cycles by measuring serum E2 and P4 levels on D2 and at the time of hCG trigger. The dose of gonadotropins dose should be chosen in such a way that the number of oocytes retrieved should not be more than 10 to 12. When E2 and P4 are elevated over a threshold value, one should cryopreserve the embryo and transfer them in the subsequent cycle to decrease the incidence of ovarian hyperstimulation syndrome and improve the IR.

Keywords: Assisted reproductive technology outcome, clinical pregnancy rate, and utilization rate, embryogenesis, implantation rate, non-polycystic ovary syndrome, polycystic ovary syndrome

How to cite this article:
Patil M, Reddy P, Chandrashekar C, Patil M. Is embryogenesis and ART outcome different in polycystic ovary syndrome?. Onco Fertil J 2019;2:79-86

How to cite this URL:
Patil M, Reddy P, Chandrashekar C, Patil M. Is embryogenesis and ART outcome different in polycystic ovary syndrome?. Onco Fertil J [serial online] 2019 [cited 2023 Mar 29];2:79-86. Available from: https://www.tofjonline.org/text.asp?2019/2/2/79/277445

Introduction

Polycystic ovary syndrome (PCOS) is a common endocrine metabolic dysfunction, affecting 5%–10% of women of reproductive age.^[1] The etiology of the syndrome is still unknown. The European Society of Human Reproduction and Embryology and the American Society for Reproductive Medicine defined the criteria for the diagnosis of PCOS at a meeting held at Rotterdam in 2003.^[2],[3] PCOS is traditionally defined by the Rotterdam criteria which include two of the three criteria which include polycystic ovaries, biochemical or clinical signs of androgen excess and ovulatory dysfunction.^[2],[3] Insulin resistance is common in both lean and obese PCOS women. Hyperinsulinemia due to insulin resistance occurs in up to 80% of women with PCOS and central obesity and 30%–40% of lean PCOS women.^[4],[5] The endocrine–metabolic disturbances in women with PCOS may result in abnormal ovarian function, resulting in abnormal folliculogenesis, steroidogenesis, and lack of development of a dominant follicle.^[6]

The oocyte maturation may also be affected^[7] and granulosa cell (GC) function is abnormal, particularly in anovulatory women with PCOS. Follicles from women with PCOS are more heterogeneous than those from normal ovaries and include a significant subpopulation that hypersecrete both oestradiol and progesterone.^[8] PCOS is also associated with hypersecretion of luteinizing hormone (LH). Impaired oocyte maturation and embryonic developmental competence in PCOS women is possibly linked with abnormal endocrine/paracrine factors, metabolic dysfunction, and alterations in the intrafollicular microenvironment during folliculogenesis and follicle maturation.^[9] At in vitro fertilization (IVF), although the number of oocytes retrieved is more, it is often associated with poor oocyte quality, lower fertilization and cleavage rate (CR), implantation rate (IR), and higher miscarriage rate.^{[10],[11],[12],[13]}

Aim

To compare the embryogenesis and outcome of assisted reproductive technology (ART) in PCOS and non-PCOS women, who were classified depending on the estradiol levels on the day of human chorionic gonadotropin (hCG)
To determine whether embryo quality, IR, and clinical pregnancy rate (CPR) are related to estradiol (E2) or progesterone (P4) levels on the day of hCG.

Materials and Methods

This was a retrospective cohort study of 425 women of which 183 were PCOS and 242 were non-PCOS who were matched for body mass index and age. We compared the follicle stimulating hormone (FSH), antral follicle count (AFC), anti-Mullerian hormone (AMH), thyroid-stimulating hormone, total dose of gonadotropin used, total days of stimulation, estradiol (E2), and progesterone (P4) on the day of hCG trigger between both groups. Before entering the IVF program, patients were classified into PCOS and non-PCOS depending on the AFC at the baseline scan done on day 2/3 of the menstrual cycle (MC). We calculated the fertilization, cleavage, blastocyst formation rate, and utilization rate of the embryos from the total oocytes retrieved and fertilized in four different groups classified according to the E2 levels on the day of the hCG (≤1000, 1001–2000, 2001–3000, >3000 pg/ml) in both PCOS and non-PCOS women. We also looked at the IR and CPR in the above four groups in fresh cycle and included the frozen embryo transfer cycles to calculate the utilization rate. Statistical comparison was done using the Mann–Whitney U test.

Inclusion criteria at ultrasonography for polycystic ovary syndrome

Ovarian volume >10 ml
The presence of microcyst (2–8 mm diameter) around the cortex (at least 15)
Increased stroma.

Exclusion criteria

Patients with incomplete data
Patients with cycle cancellation.

In vitro fertilization protocol

We used either Rec. FSH or combination of Rec. FSH and human menopausal gonadotropin for COS
Gonadotropin-releasing hormone (GnRH) antagonist was given to prevent premature LH rise
Rec. hCG was administered for trigger when there were at least 3 follicles >17 mm in diameter
Estradiol and progesterone levels were done on day 2 of the MC and on the day of hCG in all patients
Estradiol level was also done on day 5 of MC or day 4 of COS in all patients
Estradiol levels were repeated only in the presence of poor response or hyper response
Oocyte retrieval was done 35 h after ovulation trigger.

The presence of fertilization confirmed by the presence of 2 pronuclei (PN) or syngamy at 18 h post insemination or sperm injection. Cleavage was looked for at 48 h, where the presence of 4 cells is optimal development. Embryogenesis was followed to look for 8 cell embryos on day 3 and blastocyst on day 5 or 6. Grades of the embryo were noted on day 2, 3, 5, and 6. Grading of cleavage cell embryos was done depending on symmetry and number of blastomeres, amount of anucleate fragmentation, and number of nuclei in each blastomere. Embryos were classified into 12 groups from Grade 1A to Grade 4C, where 1A being the best.

The numbers and letters represent the following:

Blastomere uniform in size and shape and little or no fragmentation
Blastomeres uneven in size and shape and/or fragmentation <10% of the embryonic surface
Fragmentation 10% ±30% of the embryonic surface
Fragmentation >30% of the embryonic surface.

The numbers and letters represent the following:

Embryos having only mononucleated blastomeres
Embryos having one or more blastomeres containing no visible nucleus
Embryos having one or more multinucleated blastomeres.

Scoring of day 3 embryos

Grade 1 = 8-cells, <10% fragmentation, good cell–cell contact, no multinucleated blastomeres
Grade 2 = 8-cell, 10%–20% fragmentation or lacking good cell–cell contact, no multinucleated blastomeres
Grade 3 = 6–7-cell or 8-cell with 20% fragmentation or uneven blastomere size, no multinucleated blastomeres
Grade 4 = Greater than 8-cell or 4–6-cell or 8-cell with >20% fragmentation or uneven blastomere size or multinucleated blastomeres
Grade 5 = Less than 4-cell or grossly fragmented or with half of the blastomeres being multinucleated.

Whereas on day 5 embryos (blastocyst), were graded depending on the degree of expansion, number of trophoectoderm (TE) cells, cohesiveness and size of TE cells, number of cells, and compaction of inner cell mass (ICM) (Gardner and Schoolcraft, 1999; Gardner et al., 2000).^[14],[15]

Day 5/6

Gardner and Schoolcraft blastocyst morphology grading

Given a numerical score from 1 to 6 on the basis of their degree of expansion and hatching status, as follows:

Early blastocyst with a blastocoel that is less than half of the volume of the embryo
Blastocyst with a blastocoel that is half of or greater than half of the volume of the embryo
Full blastocyst with a blastocoel completely filling the embryo
An expanded blastocyst with a blastocoel volume larger than that of the early embryo, with zona thinning
Hatching blastocyst with the TE starting to herniate through the zona
Hatched blastocyst, in which the blastocyst has completely escaped from the zona.

The development of the ICM was graded as follows:

Tightly packed, many cells
Loosely grouped, several cells
Very few cells.

TE was assessed as follows:

Many cells forming a cohesive epithelium
Few cells forming a loose epithelium
Very few large cells.

The primary outcome measured was IR and CPR, whereas the secondary outcome measured was fertilization rate (FR), CR blastulation rate, embryo quality, and utilization rates.

Mann–Whitney U test and Chi-square test were used for statistical analysis.

Results

The AFC and AMH were significantly higher in the PCOS group (P = 0.001). More number of patients in the non-PCOS group required the addition of LH (P < 0.001). The total dose of gonadotropins required in the non-PCOS was also higher; however, there was no difference in the days of stimulation. The P4 level was similar in both the groups, but the E2 was significantly higher in the PCOS group [Table 1]. There was no statistical significance in the FR (P = 0.563), CR (P = 0.951), but the blastocyst formation rate was significantly higher in the non-PCOS group.

Table 1: Clinical characteristic

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FR was significantly higher in PCOS group when the E2 was between 2001 and 3000 pg/ml when compared to non-PCOS group (P < 0.001). The FR was significantly higher in the PCOS group when the E2 levels were between 2001 - 3000 pg/ml. There was no difference in the FR in the other groups [Table 2]. There was no difference in CR across the E2 groups [Table 3]. The blastocyst formation rate was significantly higher in non-PCOS compared to PCOS group when E2 level was more than 2000 pg/ml [Table 4]. There was no statistical difference seen in the grade of the embryos on day 3 and day 5 across the E2 groups in the PCOS women. In the non-PCOS, high E2 did not affect the embryo quality on D3 and D5 and the number of Grade 1 embryos was similar across the four E2 groups [Table 5] and [Table 6]. The quality of embryos on day 3 and day 5 was much better in non-PCOS group with E2 levels >2000 [Table 5] and [Table 6].

Table 2: Fertilization rate

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Table 3: Cleavage rate

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Table 4: Blastocyst formation rate

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Table 5: E2 level and grade of embryo on day 3

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Table 6: E2 level and grade of embryo on day 5

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The utilization rate per egg retrieved [Table 7] was significantly higher in PCOS group when E2 was <1000 pg/ml (P = 0.00013) whereas it is significantly higher in non-PCOS patients when estradiol was more than 3000 pg/ml group (P =0.022). The utilization rate of the embryos per egg retrieved when compared between the PCOS and non-PCOS patients, it was significantly higher in non-PCOS when E2 was more than 2000 pg/ml (P = 0.004 when E2 2001–3000 pg/ml and <0.001 when E2 >3000 pg/ml). The utilization rate of the embryos per egg retrieved and was significantly higher in PCOS patients as compared to non-PCOS when E2 was <1000 pg/ml (P = 0.012).

Table 7: Correlation of E2 levels with utilization rate, clinical pregnancy rate, and implantation rate

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The utilization rate per 2PN was similar across the groups in the non-PCOS group (P = 0.327), whereas it was significantly higher in the PCOS group when E2 was <1000 pg/ml (P = 0.00003). When the E2 was more than 2000 pg/ml, the utilization rate per 2PN was significantly higher in the non-PCOS group(P < 0.001 when E2 2001–3000 pg/ml and >3000 pg/ml). The utilization rate per 2PN was significantly higher in the PCOS group when E2 was1000 pg/ml (P = 0.010).

No difference in the CPR across the E2 groups non-PCOS patients. The CPR was significantly higher when E2 values were between 2001 and 3000 in PCOS group (P = 0.0007). There was no difference in the pregnancy rates when PCOS and non-PCOS patients were compared across the four E2 groups[Table 8].

Table 8: E2 levels and clinical pregnancy rate in polycystic ovary syndrome and nonpolycystic ovary syndrome patients

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The IR was similar across the four E2 groups in the non-PCOS patients (P = 0.919). In the PCOS group, it was highest when E2 was between 2001 and 3000 pg/ml (P = 0.00075) suggesting that optimal E2 levels are necessary for implantation. The IR was higher in the PCOS group as compared to non-PCOS in women with E2 between 2000 and 3000 (P = 0.020) [Table 9].

Table 9: E2 levels and implantation rate in polycystic ovary syndrome and nonpolycystic ovary syndrome patients

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CPR in fresh cycles was significantly higher in the PCOS group (42.27%) than the non-PCOS group (33.69%). The cumulative pregnancy rate was higher in the PCOS group as a more number of embryos were frozen (39.41% vs. 31.17%). The abortion rate was 20% both in the PCOS and non-PCOS group. There were 4 ectopic pregnancies in the PCOS group and 1 in the non-PCOS group.

Embryo transfer was not done in 13 women in the PCOS group and 11 women in the non-PCOS group.

Discussion

Human oocyte and the resulting embryo are the most important variables for the success of IVF. Oocyte quality will be dictated by events that occur during both growth and maturation stages of development, as oogenesis is continuum of months.

It is influenced by intrinsic factors such as age and genetics or extrinsic factors such as stimulation protocols, culture conditions, and nutrition.

IVF involves the collection of oocytes which are exposed “in vivo” to higher level of FSH and estradiol in the past 14 days of folliculogenesis and growth, GnRH agonist in the past 30 days of growth and GnRH antagonist in the past 7 days of growth.

It is not known if oocytes obtained are similar to “natural cycle” oocytes though they can result in live births. Only about 5% of oocytes collected and used the result in live births in patients aged under 37 years and this decreases to 1%–2% in women aged over 40 years.^[16]

The cause for this though not known but could be related to controlled ovarian stimulation (COS) as it interferes with the balance of forces within the ovary, overrides their endogenous pattern of control which optimize normal selection process and can affect the cytoplasmic maturity and result in cytoplasmic and spindle defects. Therefore, before starting ovarian stimulation, it is important to analyze the ovarian reserve, define the goal of ovarian stimulation and select the correct stimulation protocol.

Ovarian reserve markers such as AFC and AMH predict response to tailor the correct stimulation regimen for adequate response so as to prevent complications and improve pregnancy outcomes. It is important to know the optimum quality and quantity of oocyte/embryo required for optimal IVF outcome. Features of the oocytes one need to look at are:

Whether it is competent to undergo fertilization, chromatin remodeling, and DNA repair
Can achieve both nuclear and cytoplasmic maturity to sustain embryonic development and support timely completion of cleavage divisions
Can reliably segregates chromosomes with proper spindle formation and checkpoint functions
Activate the embryonic genome at 8-cell stage with chromatin remodeling and establishment of genomic imprinting defects.

The two parameters used to measure the success of ART are ongoing pregnancy and live birth per cycle/per embryo transfer.^[17] However, the cumulative live birth rate per cycle is a better measure of the biological potential of a cohort of oocytes.^[18],[19]

In our study, apart from CPR and IR, we looked at the utilization rate per oocyte retrieved and fertilized. These calculations are useful to elucidate the overall efficiency/inefficiency of an ART cycle and also provide detailed information about the competence of the gametes and embryos and thereby offering more realistic expectations for a given cycle.^[20]

Several publications have shown that minimal stimulation gives optimum results as more number of oocytes does not necessarily mean a higher utilization rate per oocyte cohort retrieved in one cycle.^[2],[6],[21]

In our study, the utilization rate was significantly higher in non-PCOS patients when estradiol was more than 3000 pg/ml group whereas it was significantly higher in PCOS group when E2 was <1000 pg/ml. This suggests that it is not only the estradiol levels which will influence the utilization rate but probably the oocyte quality is also responsible. These results suggest that PCOS can also affect the oocyte quality apart from embryo-endometrial asynchrony. This is also evident from the fact that the utilization rate in the non-PCOS patients was significantly higher when the estradiol levels were more than 2000 pg/ml. When we looked at the utilization rate per 2PN that is per oocyte fertilized, there was no difference in the non-PCOS patients. In PCOS patients, the utilization rate per 2PN was significantly higher when estradiol levels were <1000 pg/ml, which reinforces the fact that PCOS can also affect the oocyte and embryo quality, though morphologically day 5 embryos were similar across all the four estradiol groups. If the embryo quality was compared between the PCOS and non-PCOS patients, it was significantly better in the non-PCOS patients especially when estradiol levels were more than 2000 pg/ml. Probably, it is not only the morphology of the oocyte and embryo that is important for implantation, the biological competence is equally important. In the present study, we reported a total utilization rate per oocyte retrieval and per 2PN of 29.54% and 38.74% in PCOS patients and 36.44% and 49.72% in non-PCOS patients, respectively. The utilization rate was thus much better in the non-PCOS patients as compared to PCOS patients.

In PCOS women impaired oocyte maturation and embryonic developmental competence is possibly linked with abnormal endocrine/paracrine factors, metabolic dysfunction and alterations in the intrafollicular microenvironment during folliculogenesis and follicle maturation.^{[9],[22],[23],[24]} In PCOS extra- and intra-ovarian factor can impact on GC– oocyte interactions, oocyte maturation, and potential embryonic developmental competence. In PCOS, deficiency of FSH, hypersecretion of LH, hyperandrogenemia, hyperinsulinemia, and altered growth factors and cytokines can influence the oocyte and embryo competence. These factors may cause abnormalities during folliculogeneis, follicular growth, and oocyte meiotic maturation processes.^[11] Although we presume that the extra- and intra-ovarian factors can affect the developmental potential of the oocytes and embryos, it is difficult to elucidate the molecular mechanism involved.^[11]

The live birth rate per patient recruited was 39.41% in the PCOS and 31.17% in the non-PCOS, suggesting a higher cumulative pregnancy rate in PCOS women as more number of embryos could be frozen. This signifies that with optimal COS protocols where the estradiol levels are maintained below 2500 pg/ml and the number of oocytes obtained to <12–14, one can have an optimal ART outcome. This was also evident from the fact that the IRs were best when estradiol levels were more than 2000 pg/ml and <3000 pg/ml.

Therefore, systematic screening for ovarian reserve and key factors, especially in PCOS women like AMH will help in selecting the best COS protocol to effectively improve oocyte maturation and developmental competency for an optimal ART outcome with increased live birth rate is essential.

Conclusion

We have seen that only a small part of the oocytes retrieved after assisted reproduction has the competence to develop into a live birth of a child. This may be related to the oocyte and embryo competence as well as endometrial receptivity. As the embryogenesis and endometrial receptivity can be affected by high estradiol and progesterone levels it is highly recommended to closely monitor controlled ovarian stimulation cycles by measuring serum estradiol and progesterone levels on day two of MC and on the day of trigger for an optimal outcome. We can maximize the success rates by stimulation individualization and with freeze all and embryo transfer in a subsequent natural or hormone-stimulated cycle to improve the implantation and live birth rate.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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