Low Gonadotropin Dosage Reduces Aneuploidy in Human Preimplantation Embryos: First Clinical Study in a UAE Population

Abstract

Type of Study:

Retrospective analysis of embryo aneuploidy in patients undergoing in vitro fertilization (IVF) cycles.

Aim:

To evaluate factors that might affect the incidence of embryo aneuploidy during IVF cycles.

Methods:

Three hundred twelve IVF cases were included in the present study. Preimplantation genetic testing for aneuploidy (PGT-A) was performed for all the subjects involved. Subject stratification was done based on maternal age, gonadotropin drug dosage, and IVF outcomes data. Maternal age <35 years were placed in the “Young” age group and age ≥35 years were placed in the “Advanced Maternal Age” group. Similarly, IVF drug administered <200 International units (IU) was considered “low dosage,” group and ≥200 IU were considered “high dosage” group. Patients were stratified into four groups—group 1: age <35 years and administered <200 IU; group 2: age <35 years and administered ≥200 IU; group 3: age ≥35 years and administered at <200 IU; and group 4: age ≥35 years and administered ≥200 IU. PGT-A results were attained using a next-generation sequencing-based protocol. Embryo transfer was guided by transabdominal ultrasound. Statistical significance was calculated with the use of chi-square test.

Results:

One thousand fifty blastocyst trophectoderm biopsies from 312 IVF cases were retrieved. The IVF outcome of a total of 105 normal cases resulted in 65.71% pregnancies. Stratifying for maternal age and IVF drug stimulation with PGT-A analyses we found the euploid embryo percentages equal to 37.59% in Group 1; 16.18% in Group 2; 22.44% in Group 3; and 2.59% in Group 4. Similarly the aneuploid embryo (percentage)s were 62.40% for Group 1; 83.81% for Group 2; 77.55% for Group 3; and 87.40% for Group 4.

Conclusion:

This is the first clinical study reporting that gonadotropin dosage may act as a contributing factor in increasing aneuploidy incidences for the patients undergoing IVF cycles in the UAE population. This study shows that in all patient age groups, lower drug stimulation leads to an increasing trend in embryo euploidy.

Introduction

The availability of viable embryos with high implantation potential is the most important thing to tackle for efficacious in vitro fertilization (IVF). Even though high-grade embryos may be accessible for transfer, only a small percentage of patients undergoing IVF will ever achieve a pregnancy (Braga et al., 2012). High chromosomal abnormalities incidence in oocytes and embryos contributes to low fertility rate (Gianaroli et al., 1999; Munné et al., 2003). Aneuploidy incidence in human oocytes has been reported to range from 15.0% to 20.0% (Haaf et al., 2009). Up to 30% of early embryos fail to develop into normal fetuses (Wilcox et al., 1988).

The occurrence of embryo aneuploidy increases with maternal age (Hassold and Hunt, 2001). In advanced maternal age (AMA) patients, the availability of transferable embryos was affected by the low quantity and quality of oocytes retrieved after ovarian stimulation, which is closely related to the ovarian reserve (Broekmans et al., 2007).

Embryos aneuploidy may also be affected by ovarian stimulation regimens employed in IVF (Munné et al., 1997; Katz-Jaffe et al., 2005). Studies show where milder ovarian stimulation for IVF was associated with a reduced human preimplantation embryo aneuploidy rate (Baart et al., 2007). Oocytes or embryos may be affected by the high concentration of circulating steroids which is because of exogenous administration of gonadotrophins used for ovarian stimulation (Hansen et al., 2002).

The early stage of the human embryo derived from IVF treatment, chromosome aberrations, are most common (Harper et al., 1995; Munné and Cohen, 1998). However, animal studies seem to support an association between follicle-stimulating hormone (FSH) exposure and embryonic aneuploidy (Elbling and Colot, 1985; Sato and Marrs, 1986; Edgar et al., 1987), whereas human studies have been more conflicting (Munné et al., 1997; Roberts et al., 2005; Massie et al., 2008, 2011; Verpoest et al., 2008). Possible adverse effect of ovarian stimulation on oocyte and embryo quality is still the subject of lively debate. Until now, except for AMA, there are no clearly established factors for the occurrence of aneuploidy incidences.

In this study, we aim to clinically evaluate the effect of ovarian stimulation that might be related to the embryo aneuploidy incidence in patients undergoing IVF-preimplantation genetic testing for aneuploidy (PGT-A) cycles.

Materials and Methods

Experiment design

All IVF-PGT-A cycle data were obtained between the years 2015 and 2017 at Al Ain fertility Center, Abu Dhabi, UAE. Study population consists of 100% Emirates as ethnicity percentage with patients between 19 and 39 years of age, repeated implantation failure, and/or “AMA” are the only referral indications for PGT-A. Patient's ovarian reserve status is not referred as “poor responder,” as it does not fall under the Bologna criteria (Ferraretti et al., 2011). Dosage ≥200 International units (IU) is only given to patients having minimum borderline ovarian reserve test cutoff values, to avoid the risk of inadequate response to low ovarian stimulation. Study assessment includes embryo euploidy-aneuploidy status and maternal age with low-high drug stimulation index. Group limits were set, maternal age <35 years considered “Young,” and age ≥35 years were AMA and are considered under inclusion criteria (Rubio et al., 2010; Baker et al., 2015; Department of Health-Abu Dhabi 2018). Similarly, IVF drug administered <200 IU was considered “low dosage,” and drug administered ≥200 IU was “high dosage” (Sterrenburg et al., 2011). Patients were divided into four groups—group 1 (67 cases): age <35 years and administered <200 IU; group 2 (87 cases): age <35 years and administered ≥200 IU; group 3 (18 cases): age ≥35 years and administered <200 IU; and group 4 (140 cases): age ≥35 years and administered ≥200 IU. Embryo transfer strategies include transabdominal ultrasound (TA-US)-guided embryo transfer and further correlated with IVF outcomes.

Statistical significance calculated with the use of chi-square test (GraphPad Prism version 7.00). All statistical analyses were two-sided and p < 0.05 was considered to be statistically significant.

Consent approval

Informed written consent was obtained from all participants.

PGT-A whole-genome amplification and next-generation sequencing platform

Trophectoderm biopsies from 312 IVF cases were retrieved, and whole-genome amplification (WGA) was performed using the Ion SingleSeq™ Kit amplification system. Furthermore, these amplified WGA products were processed by next-generation sequencing (NGS) (Ion Reproseq PGS Kit).

Results

In the present study, successful results were attained from 312 out of 312 cases. Out of 1050 embryos, 612 (58.3%) are in the young group, and 438 (41.7%) fall in AMA groups.

Out of 312 IVF cases, 154 were “Young,” and 158 were “AMA.” Group 1 (67 cases): 266 embryos were retrieved, in which 100 (37.59%) embryos were normal and 166 (62.4%) embryos were abnormal; group 2 (87 cases): 346 embryos were retrieved, in which 56 (16.18%) embryos were normal and 290 (83.81%) embryos were abnormal; group 3 (18 cases): 49 embryos were retrieved, in which 11 (22.44%) embryos were normal, and 38 (77.55%) embryos were abnormal; and group 4 (140 cases): 389 embryos were retrieved, in which 49 (12.59%) embryos were normal, and 340 (87.4%) embryos were abnormal. On comparing normal-abnormal embryo between group 1 and 2, it was found to be statistically significant (p-value = 0.0001); however, between group 3 and 4 it was nonsignificant (p-value = 0.0587) (Figs. 1 and 2).

FIG. 1.

Statistical analysis of group 1-2 with normal-abnormal embryos.

FIG. 2.

Statistical analysis of group 3-4 with normal-abnormal embryos.

Embryos were transferred in young and AMA patients; we had normal cases of 70 (156 embryos) and 35 (60 embryos), respectively. IVF outcome of total 105 normal cases results in 69 (65.71%) pregnancies, of which young patient accounts for 51 (48.57%) pregnancies, and AMA accounts for 18 (17.14%) (Table 1).

Table 1.

In Vitro Fertilization Outcomes Case Number (Percentage) Stratified by Patient Age Group

Patient age group	IVF outcomes
	Normal embryo	Pregnancy	Nonpregnancy
	cases (%)	cases (%)	cases (%)
Young age (Age <35 years)	70 (66.67%)	51 (48.57%)	19 (18.10%)
Advanced maternal age (Age ≥35 years)	35 (33.33%)	18 (17.14%)	17 (16.19%)
Total IVF outcome (pregnancy and nonpregnancy)		69 (65.71%)	36 (34.29%)

IVF, in vitro fertilization.

Discussion

Studies suggest that mild ovarian stimulation leading to a lower oocyte yield has been associated with lower embryo aneuploidy and are linked with a higher chance of conceiving (Hohmann et al., 2003; Baart et al., 2007; Verberg et al., 2009). Mild stimulation also results in high-grade embryos for transfer, as indicated by good embryo morphology, and pregnancy rates comparable to those following high ovarian stimulation (Hohmann et al., 2003). In another meta-analysis study, Sterrenburg et al. (2011) have shown that 150 IU/day dose was associated with a slightly lower oocyte yield, but when compared with 200 IU/day doses, similar embryo cryopreservation and pregnancy rates were observed. The risk of insufficient response to ovarian stimulation was highest in the 100 IU/day dose group. In the present study, to avoid the risk of inadequate response to low ovarian stimulation, a 200 IU/day dose cutoff was set. Similarly, Maternal age <35 years was considered “Young” and age ≥35 years was “AMA” (Rubio et al., 2010; Baker et al., 2015; Department of Health-Abu Dhabi, 2018).

In addition to this, we also clinically evaluated the effect of IVF drug stimulation as a parameter for embryo aneuploidy incidences in the UAE population. A total of 1050 embryos were analyzed from 312 cases. Higher embryo euploidy was observed in patients administered with a low gonadotropin dosage. By comparing the dosage effect in groups 1 and 2, it was found to be statistically significant, which supports dosage role in aneuploidy incidences, while there was no statistically significant difference observed in groups 3 and 4. Nevertheless, comparing group 3 and 4, we observe a trend as group 3 euploidy percentage is more than that of group 4 (Figs. 1 and 2).

Embryo transfer strategy in our center follows TA-US-guided embryo transfer, which results in 65.71% pregnancies. Larue et al. (2017), show that embryo transfer guidance performed under transvaginal over TA-US, results in increased pregnancy in the general population (38%, vs. 30%) and the reference population age was <38 years (45% vs. 36%). However, Cozzolino et al. (2018) did not find significant differences between transabdominal-vaginal ultrasound-guided embryo transfers. Furthermore, larger randomized controlled trials required to explore the possible benefits of different embryo transfer techniques.

The finding from the present study is also consistent with the results of Baart et al. (2007); the author observed 71/143 (50%) normal embryos in the mild stimulation group (150 IU) and 61/159 (38%) chromosomally normal embryos in the conventional stimulation (225 IU). In a crossover study of Rubio et al. (2010), in which 22 oocyte donors, age between 18 and 35 years, underwent controlled ovarian stimulation in a gonadotropin-releasing hormone agonist downregulation protocol. The chromosome abnormality percentages were 65.5% with the standard protocol (225 IU) and 50.9% with reduced doses (150 IU). Another study by Baker et al. (2015), examined a total of 658,519 fresh autologous cycles of IVF correlated total gonadotropin dose and live birth rate. Cycles categorized by oocytes retrieved, FSH dose, and female age (<35, 35-39, and ≥40 years). The overall study shows that an increase in FSH dose leads to a significant decrease in live birth rate.

Conventional higher ovarian stimulation practices for IVF appear to impair oocyte quality and embryo aneuploidy in a dose-dependent manner. There is supportive evidence for having potentially adverse effect of supraphysiologic steroid levels on oocyte and embryo quality (Valbuena et al., 2001; Baart et al., 2007), corpus luteum function (Fauser and Devroey, 2003; Beckers et al., 2006), and on endometrial receptivity (Simón et al., 1995; Devroey et al., 2004). Furthermore, studies also suggested that oocyte yield after superovulation is associated with increased maternal meiotic segregation errors (Haaf et al., 2009). However, an increased risk of miscarriage and fetal aneuploidy has been reported in IVF cycles with a high response or even hyperstimulation syndrome (Nasseri et al., 1999; Raziel et al., 2002; O'Brien et al., 2009).

The present study involves strong plus points, as it does not deal with the donor selection process, so there is no conflict as far as the patient ethnic origin is concerned. Second, we analyze trophectoderm biopsies, which give more precise results than day 3 biopsies (Thompson et al., 2013). also In addition, our results are also significantly correlating with other quite similar studies performed by groups with different data sets. A possible weakness is lack of population diversity, which may limit the widespread applicability of the results. Also, in the current study, we have not included the circulating steroids level. Therefore, we are unable to state biological plausibility between high circulating steroids leading to abnormal oocyte quality. Finally, while comparing euploidy levels between group 3 and 4, group 3 shows an increasing trend in embryo euploidy, but has not reached a statistically significant level. This may be attributed to the lower sample size. Therefore, more studies are warranted in different ethics and social communities and more number of samples to further strengthen these findings.

Consequently, this study model supports the application of lower ovarian stimulation dosage for patients undergoing IVF-PGT-A cycle. In conclusion, this is the first clinical study reporting gonadotropin dosage as a contributing factor involved in euploidy incidences for IVF patients in the UAE population.

Also, our findings need to be endorsed by other groups, as both treatment strategies and PGT-A procedures vary between centers. We suggest that future IVF drug stimulation approaches may aim at engendering a sufficient number of chromosomally normal embryos and avoid boosting oocyte yield, by reducing interference with ovarian physiology, thus reducing embryo aneuploidy incidence, and enabling selection of the best-quality embryo for transfer.

Footnotes

Authors' Contributions

K.S.: study design, execution of the project work, interpretation of data and results, preparation of the article, critically revised the article, and approved the submitted versions; D.U., R.D., and M.M.V.: guidance for the laboratory work, data, and interpretation of results and preparation of the article; F.A., R.A., and L.B.: embryo biopsy and tubing; M.S. S.S.: support in the enrollment of patients in the study, patient referral for molecular studies; B.P.: support in the enrollment of patients in the study, clinical documentation for the patient's and proforma filling, and patient referral for molecular studies and reviewing of the article.

Acknowledgments

The authors thank the doctors for their assistance in patient enrollment; Dr. S. Aldayeh for technical advice; and colleague, Mr. M. Alhayek, for his time and valuable assistance. This study was funded by Al Ain Fertility Center, Al Ain UAE.

Author Disclosure Statement

No competing financial interests exist.

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