Principles and Practice of Controlled Ovarian Stimulation in ART 1st ed.

36. Effect of Ovarian Stimulation Protocols on Oocyte and Embryo Quality

James Catt 


Optimal IVF, 4234, Black Rock North Melbourne, VIC, 3195, Australia

James Catt



A review of oocyte and embryo quality suggested that the usual parameters of pregnancy are not sensitive enough to determine differences possibly caused by ovarian stimulation. The dual methodology of time to syngamy and appropriate on time embryo development was investigated to determine whether they would be useful methodology for oocyte and embryo quality. We could determine no differences between pituitary suppression, follicular stimulation, and follicular maturation with “standard” stimulation protocols for “normal” responders. Differences were found with “out of protocol” stimulations and possibly among adjuvants used. We contend that the syngamy early development model should be used with more conventional outcomes to help determine any benefits of new protocols.


Ovarian stimulation for IVFOocyte qualityEmbryo qualityQuantitative outcomesSyngamyAlpha/ESHRE consensus

36.1 Introduction

The potential effect of stimulation protocols can be very important in IVF as the maxim “you cannot make good embryos with bad eggs” still holds today. An extension to this maxim would be that we can make “bad” embryos from “good” oocytes using inappropriate materials and methods. In other words, embryo quality is dependent on many more variables than oocyte quality, and since this chapter is about the effects of stimulation, the discussion will be largely restricted to oocyte quality.

There are few central problems with assessing the effect of stimulation protocols on outcomes, and these include the drugs themselves, their inappropriate use in terms of dosage and duration of stimulation, or the unproven “mixing and matching” of various protocols. Another difficulty we face when assessing stimulation protocols is the objective, quantitative measurement of oocyte, and embryo quality. The literature abounds with publications investigating the efficaciousness of protocols, but the outcome measures are often unsatisfactory. In addition, the effect of stimulation on the endometrium itself has rarely been addressed in the literature. This chapter concerns the effects of stimulation on oocytes and embryos and the effects on the endometrium, and implantation is considered in other chapters in this book.

This chapter attempts to unravel some of the data concerning the effect of stimulation protocols on oocyte and embryo quality by examining quantitative outcome measures.

36.2 Oocyte Quality

To define “good” quality oocyte is, essentially, very difficult. The best quality oocytes are those which are most likely to implant, if fertilized in an appropriate fashion. The road to implantation however is dependent not solely on oocyte quality but a host of other variables inherent in fertilization, growth of a subsequent embryo, and a receptive endometrium. The number of variables is huge; well over a 100 have been identified. Two other points indicate that implantation potential of an oocyte is not a useful guide to oocyte quality. One is that the time taken for an oocyte to form an embryo, with ultrasound evidence of a fetal heart, is about 7 weeks post-oocyte retrieval which is too long a time for practitioners to make decisions about protocols to improve oocyte quality. The second point is that implantation potential only takes into account those embryos which are transferred and gives us no idea about the overall oocyte quality. What are needed are functional tests of oocytes which can be related back to overall quality.

Over the years there have been numerous publications that have claimed that various methodologies can be used to determine oocyte quality. These can be categorized into two areas, oocyte morphology/physiology and functional tests of follicular fluid and cumulus cells.

36.3 Oocyte Morphology

The morphology of collected oocytes and associated vestments can be readily viewed and subjectively measured. Such features as cumulus expansion and atresia have been suggested as markers of oocyte quality but are often used with other perceived physiopathologies such as zona pellucida defects, size of the first polar body, width of the perivitelline space, as well as cytoplasmic features such as vacuoles, refractile bodies, and cytoplasmic granulation. However, despite many observations, few pathologies [12] have been associated with poor outcomes with the exception of smooth endoplasmic reticulum condensates (SER) [34]. Even though SER condensates are reported to be deleterious, others report normal outcomes [56].

In conclusion, as scientists we attempt to relate our observations with a measurable outcome, but, in reality, these correlations often do not stand the test of time.

36.4 Follicular Functional Tests

Similar to morphology, a functional test to measure oocyte quality is attractive and has been investigated over the past decade. A review of the use of single biochemical markers and the more recent “omics” approach suggest that the methodology has promise but has not resulted in anything of clinical use, as yet [7].

36.5 Zygote Physiology

A novel functional test based on the time to the first zygote division has received little attention (except as an embryo selection tool). The time to syngamy is defined as the time from insemination (either conventional IVF or ICSI) to pronuclear membrane dissolution or division to the two-cell stage. It has been suggested by a number of investigators that the timing of syngamy is a reflection of embryo quality [89] although one recent report failed to find a correlation [10].

We believe that there is good circumstantial evidence to support the contention that entry into syngamy is indeed related to oocyte quality and by extension embryo quality. By definition, patients of advanced maternal age (>38), poor responders, and repeat implantation failure (RIF) have poor quality oocytes, and this is reflected in their rates of syngamy (Fig. 36.1). Two controls were used for this dataset, first cycle patients aged less than 39 and oocyte donors. In this graph we have plotted the percent of zygotes entering syngamy at 25 h post insemination [3]. It is clear from this graph that a much higher proportion of zygotes from first cycle patients and oocyte donors enter syngamy compared with either advanced aged (39–42) or RIF patients. Indeed the donor oocyte group shows a very high proportion of zygotes entering syngamy compared with the infertile group, possibly a reflection of their infertility.


Fig. 36.1

Percent of zygotes entering syngamy at 25 hpi for donor oocytes (42 cycles/462 zygotes), standard IVF patient <38 (94 cycles/864 zygotes), advanced maternal age >38 (76 cycles/479 zygotes), and recurrent implantation failure patients (62 cycles/484 zygotes)

For the purposes of this chapter, we will use entry into syngamy as a measure of oocyte quality.

36.6 Embryo Quality

While the maxim stated at the beginning of this chapter that only good quality oocytes can make good quality embryos is true, it is easily derailed and that we can make poor quality embryos from good quality oocytes using inappropriate conditions.

What defines a “good” embryo? There are as many embryo scoring and grading systems as there are IVF units, and some of the most commonly used criteria of fragmentation and overall morphology are currently being reexamined in the light of several observations, originally based on specific observation times and, more recently, the advent of time lapse recording systems [3]. Even with the most stringent scoring systems, the “best” embryos have only a 50 % chance of implantation. This suggests that the use of such systems is limited as to its efficacy. More recently, the term “on time, appropriate development,” as defined by the Alpha/ESHRE consensus, has proved to be at least as effective as the more complex methods. Again, time lapse measurements of early development are adding more precise information to appropriate development [11].

On time, appropriate development and the effect on implantation rates are illustrated in Fig. 36.2, where the same dataset used for Fig. 36.1 was used. It is quite clear from this graph that the embryos from the poorer prognosis groups develop at a slower rate, and these differences increase with extended culture.


Fig. 36.2

The percentage of zygotes from each group meeting their developmental milestones. These milestones were 4-cell embryos at 43 h post insemination (hpi) on day 2, >7 cells at 67 hpi for day 3, and blastocoel formation at 115 hpi for day 5

The differences between the groups are further illustrated in Fig. 36.3, where the ratio of blastocysts forming on day 5 was greatest for the “good” prognosis groups (donor and first cycle patients) compared with the “poor” prognosis groups (advanced maternal age and RIF patients). The overall utilization rates (defined as the percent of zygotes that were either transferred or frozen) and implantation rates per embryos transferred fresh follow the same trends. The apparent decrease in miscarriage rates suggests that “good” prognosis embryos are less liable to fail in pregnancy maintenance.


Fig. 36.3

The ratio of blastocysts forming on day compared with day 6 (d5:d6), the percent of zygotes that were transferred or frozen (utilized, ut), the implantation rate (measured as a fetal heart) per embryo that was transferred fresh (Ir), and the miscarriage rate after fetal heart detection

Using the “on time, appropriate development” methods should now give us more quantitative methods to look at the effect of stimulation regimes on embryonic development. However, can we make the contention that if only “good” oocytes make “good” embryos, will the measurement of zygote syngamy be sufficient by itself to investigate differences in stimulation regimens?

36.7 Evidence

There are three main areas of ovarian stimulation which can potentially affect the quality of oocytes and embryos. These are suppression of pituitary functions using GNRH agonists and antagonists, stimulation of follicular development with FSH, and the “trigger” used to initiate oocyte maturation and release. Various adjuvants have also been used to attempt to mediate all of these processes.

36.7.1 Pituitary Suppression

Data for agonist and antagonist pituitary suppression have been extensively studied, and the data is consistent with little or no difference between their outcomes when using relatively non-quantitative outcome measures such as live births [1213]. We have used syngamy and early embryonic development to compare agonist and antagonist for a substantial number of cycles in several IVF units, both in Australia and other countries. The results are summarized in Table 36.1. No statistical difference could be found with the agonist versus antagonist using non-inferiority testing with a statistical power of 95 % (>4000 zygotes in each arm). The data adds further evidence that, under normal “standard” protocols, there are no statistical differences between agonist and antagonist pituitary suppression.

Table 36.1

Comparison of early development events between agonist and antagonist pituitary suppression


Hours post insemination (hpi ± 1)

Agonist (4534 zygotes)

(% of zygotes)

Antagonist (4823 zygotes)

(% of zygotes)



58 %

59 %

>3 cell d2


68 %

66 %

>7 cell d3


50 %

54 %

Syngamy, day 2 and day 3 measurements were at the Alpha/ESHRE consensus times post insemination. Patients were aged less than 39 and having their first IVF cycle

36.7.2 Follicular Stimulation

Stimulation of follicles is usually achieved using FSH either recombinant (rFSH), extracted from urine (uFSH) or a mix of both. Again the literature is rife with reports as to the benefit of one regimen over another. It is very interesting to analyze two Cochrane reviews, the first [14] suggesting a small increase in live take-home baby rate with rFSH and the second suggesting no difference [15]. I think this underlines one of the problems using pregnancy and live birth data, as outlined above, the data having inadequate resolving power with too many confounders.

In Australia, we have not been able to use urinary-derived FSH until recently, so we have not been able to derive a comparison between urinary and recombinant products. The comparison we have been able to make is between antagonist and rFSH and antagonist and a mix of rFSH and uFSH (from an associated clinic not in Australia). The results are shown in Table 36.2. While the differences are significant (p = 0.0066), the dataset needs deriving under more stringent conditions.

Table 36.2

Comparison of early development events using either rFSH alone or using a combination of rFSH and uFSH


Hours post insemination (hpi ± 1)


(876 zygotes)

rFSH and uFSH

(642 zygotes)



56 %

64 %

>3 cell d2


65 %

70 %

>7 cell d3


47 %

49 %

Measurements and patient segmentation were as in Table 36.1

36.7.3 Follicular Maturation

Maturation of follicles has usually been achieved with hCG (either recombinant or extracted), but more recently the use of antagonist to suppress the pituitary has given us the opportunity to use agonist to mature the follicles.

The majority of studies have shown that using pregnancy rates and live births, there are no differences between the triggers between recombinant and urinary hCG [16], but there has been a small reported difference between rhCG and agonist trigger [17]. This was reported as a decrease in the pregnancy rate.

Our early development data comparing uhCG and rhCG indicated no significant differences (data not shown). There has been one report using time lapse measurement of early development comparing agonist and rhCG versus antagonist and agonist trigger [18]. There was a reported increase in syngamy rates with rhCG, but this was not reflected in subsequent divisions.

It would appear from the above discussion that stimulation protocols have little measurable effect on oocyte and early embryo quality. It is important to bear in mind that the data used to produce these results was from “good” prognosis patients stimulated with “standard” protocols. What happens when these standard protocols are not adhered to or are supplemented with adjuvants?

36.7.4 “Out of Protocol” Stimulations

There are several instances whereby “out of protocol” stimulations occur. A couple of examples of these could be “coasting” to reduce the chances of OHSS, too quick a stimulation (<7 days FSH), too slow a stimulation (>15 days FSH), and inappropriate triggering decisions with discordant follicular cohorts. One might expect that some of these circumstances could reduce oocyte and embryo quality, but substantial data has not been published. We have looked at a couple of inappropriate stimulations using our syngamy model. There was little difference with coasting for a day or two, short and long FSH duration, providing the trigger was administered appropriately (on leading follicles having a diameter of 17–19 mm). If however the trigger was delayed because of discordant follicles of a smaller diameter and the lead follicles reached >24 mm at trigger, then the syngamy and early development were negatively affected (table 36.3).

Table 36.3

Effect of delaying trigger beyond a lead follicle diameter of 24 mm


Hours post insemination (hpi ± 1)

Lead follicles <20 mm

242 cycles

Lead follicles >24 mm

42 cycles



62 %

42 %

>3 cell d2


68 %

58 %

>7 cell d3


54 %

42 %

All cycles were antagonist with rhCG trigger

36.7.5 Adjuvants

Supplements such as growth hormone, LH, Colorado protocol, DHEA, estradiol patches, and heparin have been suggested to benefit outcomes of stimulation for certain subgroups of patients, but the evidence is contradictory. The biggest problem is that the potential beneficiaries are usually few in number, and so it is difficult to get statistical data. The use of the syngamy, early development methodology outlined above, reduces the number of patients (at least fivefold) required to conduct reasonable statistics because the sample size is increased as it uses zygotes instead of patients.

The only data we currently have is in the use of the so-called “Colorado” protocol which uses a mix of antibiotic, immunosuppressant, aspirin, and estrogen during stimulation. This protocol is generally used after several failed cycles (similar to the RIF patients in Fig. 36.1). We have compared “Colorado” cycles and with the patients previous cycle without the “Colorado” protocol. The results are shown in Table 36.4. There is a suggestion that the “Colorado” is of benefit to those patients with repeated implantation failures in terms of an overall oocyte and embryo quality.

Table 36.4

Comparison between controls (cycle prior to “Colorado”) and “Colorado” cycles


Hours post insemination (hpi ± 1)

Control cycles (before “Colorado”

Colorado cycles



45 %

58 %

>3 cell d2


54 %

62 %

>7 cell d3


38 %

48 %

All cycles were agonist with uhCG trigger

36.8 Conclusions

So, is our syngamy/early cleavage model of any benefit? We believe it is for two reasons, the first being that used as a laboratory key performance indicator (KPI) one can ensure that stimulations are consistent in producing similar quality oocytes and embryos and the second is that new protocols or deviations of protocols can be monitored quickly and efficiently (as shown in the above examples). As in all investigations, the more data we have, then the more likely we will draw appropriate conclusions. Therefore, previous data as to the effects of stimulation on oocyte and embryo quality should be included in any analysis on the proviso that they are experimentally robust and statistically viable. The use of time lapse systems is proving to be invaluable as a research tool and is backing up and probably refining the use of our syngamy and early cleavage model. Whether this mandates their routine use or helps us refine our current systems remains to be seen. The take-home message from this chapter is that “standard” protocols when used correctly on those patients who respond “normally” give oocytes and embryos of equivalent quality. As always, our challenge is to broaden our range of protocols to include those who do not respond well to our “standard” ones. A quantitative estimation of oocyte and embryo quality will help us with their design.



Ebner T, Moser M, Tews G. Is oocyte morphology prognostic of embryo developmental potential after ICSI? Reprod Biomed Online. 2006;12(4):507–12.CrossRefPubMed


Setti AS, Figueira RC, Braga DP, Colturato SS, Iaconelli Jr A, Borges Jr E. Relationship between oocyte abnormal morphology and intracytoplasmic sperm injection outcomes: a meta-analysis. Eur J Obstet Gynecol Reprod Biol. 2011;159(2):364–70.CrossRefPubMed


ALPHA Scientists in Reproductive Medicine; ESHRE Special Interest Group in Embryology. Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Reprod Biomed Online. 2011;22(6):632–46.


Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod. 2011;26(6):1270–83.CrossRef


Akarsu C, Cağlar G, Vicdan K, Sözen E, Biberoğlu K. Smooth endoplasmic reticulum aggregations in all retrieved oocytes causing recurrent multiple anomalies: case report. Fertil Steril. 2009;92(4):1496.CrossRefPubMed


Mateizel I, Van Landuyt L, Tournaye H, Verheyen G. Deliveries of normal healthy babies from embryos originating from oocytes showing the presence of smooth endoplasmic reticulum aggregates. Hum Reprod. 2013;28(8):2111–7.CrossRefPubMed


Revelli A, Delle Piane L, Casano S, Molinari E, Massobrio M, Rinaudo P. Follicular fluid content and oocyte quality: from single biochemical markers to metabolomics. Reprod Biol Endocrinol. 2009;7:40.PubMedCentralCrossRefPubMed


Shoukir Y, Campana A, Farley T, Sakkas D. Early cleavage of in-vitro fertilized human embryos to the 2-cell stage: a novel indicator of embryo quality and viability. Hum Reprod. 1997;12(7):1531–6.CrossRefPubMed


Wharf E, Dimitrakopoulos A, Khalaf Y, Pickering S. Early embryo development is an indicator of implantation potential. Reprod Biomed Online. 2004;8(2):212–8.CrossRefPubMed


Lee MJ, Lee RK, Lin MH, Hwu YM. Cleavage speed and implantation potential of early-cleavage embryos in IVF or ICSI cycles. J Assist Reprod Genet. 2012;29(8):745–50.PubMedCentralCrossRefPubMed


Aguilar J, Motato Y, Escribá MJ, Ojeda M, Muñoz E, Meseguer M. The human first cell cycle: impact on implantation. Reprod Biomed Online. 2014;28(4):475–84.CrossRefPubMed


Kolibianakis EM, Collins J, Tarlatzis BC, Devroey P, Diedrich K, Griesinger G. Among patients treated for IVF with gonadotrophins and GnRH analogues, is the probability of live birth dependent on the type of analogue used? A systematic review and meta-analysis. Hum Reprod Update. 2006;12(6):651–71.CrossRefPubMed


Depalo R, Jayakrishan K, Garruti G, Totaro I, Panzarino M, Giorgino F, Selvaggi LE. GnRH agonist versus GnRH antagonist in in vitro fertilization and embryo transfer (IVF/ET). Reprod Biol Endocrinol. 2012;10:26.PubMedCentralCrossRefPubMed


Coomarasamy A, Afnan M, Cheema D, van der Veen F, Bossuyt PM, van Wely M. Urinary hMG versus recombinant FSH for controlled ovarian hyperstimulation following an agonist long down-regulation protocol in IVF or ICSI treatment: a systematic review and meta-analysis. Hum Reprod. 2008;23(2):310–5.CrossRefPubMed


van Wely M, Kwan I, Burt AL, Thomas J, Vail A, Van der Veen F, Al-Inany HG. Recombinant versus urinary gonadotrophin for ovarian stimulation in assisted reproductive technology cycles. A cochrane review. Hum Reprod Update. 2012;18(2):111–28.CrossRefPubMed


Youssef MA, Al-Inany HG, Aboulghar M, Mansour R, Abou-Setta AM. Recombinant versus urinary human chorionic gonadotrophin for final oocyte maturation triggering in IVF and ICSI cycles. Cochrane Database Syst Rev. 2011;13(4):CD003719. doi:10.​1002/​14651858.​CD003719.​pub3.


Youssef MA, Van der Veen F, Al-Inany HG, Griesinger G, Mochtar MH, van Wely M. Gonadotropin-releasing hormone agonist versus HCG for oocyte triggering in antagonist assisted reproductive technology cycles. Cochrane Database Syst Rev. 2010;10(11):CD008046. doi:10.​1002/​14651858.​CD008046.​pub2.


Muñoz M, Cruz M, Humaidan P, Garrido N, Pérez-Cano I, Meseguer M. The type of GnRH analogue used during controlled ovarian stimulation influences early embryo developmental kinetics: a time-lapse study. Eur J Obstet Gynecol Reprod Biol. 2013;168(2):167–72.CrossRefPubMed