PGT for aneuploidy does not affect three-cycle cumulative IVF discontinuation rate in women of advanced maternal age

Accepted Manuscript

Pre-implantation genetic testing for aneuploidy does not decrease three-cycle cumulative discontinuation rate when compared to conventional in-vitro fertilization in women with advanced maternal age Sezcan Mumusoglu MD , Irem Yarali Ozbek MSc, MHS , Zuhal Yapici Coskun MD , Mehtap Polat MD , Lale Karakoc Sokmensuer MD , Gurkan Bozdag MD , Hakan Yarali MD PII: DOI: Reference:

S1472-6483(19)30236-6 https://doi.org/10.1016/j.rbmo.2019.03.103 RBMO 2152

To appear in:

Reproductive BioMedicine Online

Received date: Revised date: Accepted date:

16 November 2018 16 February 2019 1 March 2019

Please cite this article as: Sezcan Mumusoglu MD , Irem Yarali Ozbek MSc, MHS , Zuhal Yapici Coskun MD , Mehtap Polat MD , Lale Karakoc Sokmensuer MD , Gurkan Bozdag MD , Hakan Yarali MD , Pre-implantation genetic testing for aneuploidy does not decrease three-cycle cumulative discontinuation rate when compared to conventional in-vitro fertilization in women with advanced maternal age, Reproductive BioMedicine Online (2019), doi: https://doi.org/10.1016/j.rbmo.2019.03.103

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ACCEPTED MANUSCRIPT Pre-implantation genetic testing for aneuploidy does not decrease three-cycle cumulative discontinuation rate when compared to conventional in-vitro fertilization in women with advanced maternal age

Sezcan MUMUSOGLU, MD1; Irem YARALI OZBEK, MSc, MHS2; Zuhal YAPICI COSKUN,

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MD2; Mehtap POLAT, MD2; Lale KARAKOC SOKMENSUER, MD3; Gurkan BOZDAG, MD1; Hakan YARALI, MD1,2

Hacettepe University School of Medicine, Department of Obstetric and Gynecology,

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Ankara, Turkey

Anatolia IVF and Women Health Centre, Ankara, Turkey

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Hacettepe University School of Medicine, Department of Histology and Embryology,

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2

Corresponding Author;

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Hakan YARALI, MD

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Ankara, Turkey

Department of Obstetrics and Gynecology

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Hacettepe University School of Medicine 06100 Ankara, Turkey.

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Tel.: +90 312 3051805 Fax: +90 312 3052835

E-mail: [email protected]

Running title: PGT-A does not decrease IVF discontinuation rate in AMA cases

ACCEPTED MANUSCRIPT Key message: Performing PGT-A did neither reduce the discontinuation rate following the 1st, or 2nd failed cycle nor the cumulative rate before completing three-cycles. Female ageing was the only independent factor to discontinue treatment with the hazard of 7% with each year of ageing.

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Abstract

Research Question: Does pre-implantation genetic testing for aneuploidy (PGT-A) influence discontinuation rate in women with advanced maternal age (AMA) undergoing

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in-vitro fertilization (IVF)?

Design: Retrospective longitudinal cohort study carried-out at a single IVF clinic in Turkey. In total, 401 consecutive AMA cases were included. Discontinuation rates of

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pre-intervention (Conventional-IVF, June 2013-October 2014; 203 couples, 270 cycles) and post-intervention (PGT-A; April 2015-June 2016; 198 couples, 285 cycles) periods

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were compared. To delineate the reason for discontinuation, phone call survey was conducted. Primary outcome measure was cumulative discontinuation rate before

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completing three-cycles of IVF treatment without achieving an ongoing pregnancy.

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Results: The discontinuation rates following 1st and 2nd failed cycles were comparable between the two arms as was the case for cumulative discontinuation rate before

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completing three-cycles. The cumulative ongoing pregnancy rate per embryo transfer was significantly higher in the PGT-A arm (42.2% vs. 16.4%, p<0.001). However, the cumulative ongoing pregnancy rate per patient was comparable between the two arms (20.7% vs. 16.2%, respectively; p=0.369).

The female age was the only significant

contributor to treatment discontinuation (HR 1.07; 95%CI, 1.09–1.13).

Of the 296

couples discontinuing treatment in both arms, 179 (179/296; 60.5%) were reached by

ACCEPTED MANUSCRIPT phone call; overall, psychological burden was the main reason for treatment discontinuation (37/179; 20.7%). Conclusions: ~ 90% of AMA cases, not achieving an ongoing pregnancy, discontinue IVF treatment before completing three-cycles. Performing PGT-A does not reduce discontinuation rate. Female ageing is the only significant contributor with the hazard of

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discontinuation from further IVF treatment by 7% with female ageing of 1-yr.

Keywords: In-vitro fertilization, pre-implantation genetic testing for aneuploidy, drop-

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out, discontinuing, advanced maternal age.

ACCEPTED MANUSCRIPT Introduction Discontinuation from in-vitro fertilization (IVF) treatment is frequent (17 – 65%) and compromises the cumulative pregnancy rates achieved with IVF (Olivius et al., 2002; Sharma et al., 2002; Verberg et al., 2008; Troude et al., 2014). The discontinuation rate varies widely from one country to another; the national policy for coverage of IVF treatment is one of the

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contributing factors for discontinuation with low rates (17%) in countries with full coverage and higher rates (64%) with partial/no coverage (Sharma et al., 2002; Dawson et al., 2005; Verberg et al., 2008; Kulkarni et al., 2014). In addition to financial concerns, advanced maternal age (AMA)

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(McDowell and Murray 2011; Troude et al., 2014), poor prognosis (Brandes et al., 2009; Troude et al., 2014), psychological and physical burden (Olivius et al., 2004; Brandes et al., 2009; Lande et al., 2015), type of ovarian stimulation (OS) protocol (Verberg et al., 2008) and medical

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disorders (Olivius et al., 2004) are among the other contributing factors for discontinuation.

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The discontinuation rate is higher in women with AMA, diminished ovarian reserve, no embryo transfer in a prior cycle and poor prognosis (Soullier et al., 2008; Brandes et al.,

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2009; McDowell and Murray 2011; Troude et al., 2014). High discontinuation rate in AMA

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and poor prognosis patients highlights the need to avoid futile cycles and maximize the livebirth rate for each treatment cycle in such patients. Therefore, a more targeted and

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technology-supported approach to treatment may be appropriate in older women to maximize the live-birth rate per embryo transfer.

Aneuploidy is the main contributor for implantation failure and increased risk of miscarriage in patients with AMA (Hodes-Wertz et al., 2012; Franasiak et al., 2014). Pre-implantation genetic testing for aneuploidy (PGT-A), by enabling the selection of euploid embryos to be

ACCEPTED MANUSCRIPT transferred, increases pregnancy and decreases miscarriage rates per transfer compared with using untested embryos (Ubaldi et al., 2015; Coates et al., 2017). In this context, PGT-A, in theory, may be expected to reduce discontinuation from IVF treatment by lowering psychological and emotional strain due to failed embryo transfer cycle in AMA cases. However, to the best of our knowledge, there is no published study evaluating the impact of

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performing PGT-A on discontinuation rates in cases with AMA, compared with conventional IVF.

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The goal of this study is to assess whether performing PGT-A influences the discontinuation rate of IVF in women with AMA compared with conventional IVF.

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Material Methods Subjects and study design

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The study is a longitudinal cohort comparison of pre-intervention (conventional IVF; June 2013-October 2014; consecutive 203 couples) and post-intervention (PGT-A with single

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frozen euploid embryo transfer (SFEET); April 2015-June 2016; consecutive 198 couples)

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periods in women with AMA undergoing IVF at private Anatolia IVF and Women’s Health Center. In-vitro fertilization treatment in both arms was completely covered by the patients

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on their own.

The only inclusion criterion was AMA (38-46 year-old); since PGT-A was offered to all couples with AMA, regardless of ovarian reserve, diminished ovarian reserve was not an exclusion criteria unless no ovarian response was noted despite maximal OS in a previous cycle with no further treatment advised by the physician. An exclusion criterion was azoospermia.

ACCEPTED MANUSCRIPT In the conventional IVF arm, all OS cycles, regardless of cycle cancellation prior to embryo transfer, were counted as a “separate cycle”. In this arm, frozen embryo replacements (FER) cycle was also counted as a “separate cycle” if there were surplus embryos to be frozen from the previous failed fresh transfer cycle. In the PGT-A arm, OS and first subsequent SFEET cycles were counted as a “separate cycle”. However, if the patient failed to conceive and had

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still surplus euploid embryo(s), then every consecutive SFEET cycle was also considered as a separate cycle, too. In both arms, cycle cancellation at the stages of OS (e.g. discordant follicular growth), egg retrieval, embryo transfer and pregnancy up to 12 weeks of gestation

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were considered as failed cycle and were included in the analysis.

The primary outcome measure was the cumulative discontinuation rate before completing

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three cycles without achieving an ongoing pregnancy. The definition of discontinuation used in the current study was not to commence a new treatment cycle, fresh or FER/SFEET, in 6

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months following a failed attempt to achieve an ongoing pregnancy. The discontinuation

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rates following the first and the second failed cycles were analyzed separately.

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Ongoing pregnancy was defined as pregnancy beyond 12 weeks of gestational age. The cumulative ongoing pregnancy rate per patient was calculated by dividing the total number

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of achieved ongoing pregnancies in three attempted IVF cycles to the number of patients in a given arm.

Treatment protocol The long GnRH agonist (Lucrin; Abott, Istanbul, Turkey) (n=248 cycles) or GnRH antagonist protocols (Cetrotide [Merck Serono, Kiel, Germany] or Orgalutran [MSD, Netherlands]) (n=

ACCEPTED MANUSCRIPT 271 cycles) were used for ovarian stimulation (OS). Depending on ovarian reserve, daily recombinant FSH (Gonal-F; Merck, Kiel, Germany) and/or highly purified hMG (Menopur; Ferring, Kiel, Germany) with initial doses of 150–450 IU/d were used for OS. After 5 days of stimulation, ovarian response was monitored with transvaginal ultrasonography and serum E2 measurements to adjust daily gonadotropin dosing. For GnRH antagonist use, fixed or

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flexible protocol was used; fixed protocol was used for patients with predicted/proven normo- or hyper-responders, whereas, flexible protocol was reserved for patients with expected/proven poor ovarian responders. Triggering of final oocyte maturation was

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performed with the use of recombinant hCG (Ovitrelle, Merck Serono, Kiel, Germany) or GnRH agonist triptorelin (Decapeptyl; Ferring, Kiel, Germany), based on the ovarian response. Oocyte retrieval was carried out under general anesthesia with the use of

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transvaginal ultrasound–guided puncture of follicles 34–36 hours after triggering final oocyte

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maturation.

After 2–4 hours of incubation, cumulus-oocyte complexes were denuded by exposure to 80

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IU/mL hyaluronidase solution diluted tenfold with G-MOPS Plus medium (Vitrolife), and also

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mechanically by plastic pipettes of defined diameters (denuding pipettes; Origio). Insemination of oocytes by means of intracytoplasmic sperm injection (ICSI) was carried out

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immediately after denudation. Each inseminated oocyte was then placed in 25 mL culture medium (G1.2 or G-TL, Vitrolife), covered by pre-equilibrated mineral oil (Ovoil; Vitrolife). Embryo culture was carried out in 6.8% CO2 and 5.0% O2.

In the conventional IVF arm, Day 3 or 5 fresh transfer was performed with surplus embryos vitrified either Day 3 or Day 5/6. Blastocyst stage fresh embryo transfer was made if there

ACCEPTED MANUSCRIPT were ≥3 good-looking embryos on day 3 assessment; day 3 embryo transfer was carried out in the remaining cases. Double embryo transfer was performed in this arm, when available. For FER cycles, artificial cycle was used.

In the PGT-A arm, trophectoderm biopsy was performed to expanding, expanded, and

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hatched blastocysts after 120–160 hours from insemination (Day 5 or 6) as previously described (Mumusoglu et al., 2017). In short, trophectoderm biopsies were sent to a reference genetic laboratory for the analysis (Genlab, Ankara, Turkey). All samples were

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processed for whole genome amplification (WGA) and array- comparative genomic hybridization (aCGH). The WGA of the biopsy samples was performed with the use of the Sureplex DNA Amplification System (BlueGnome, Cambridge, UK).

One nanogram of

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genomic DNA and one reagent-negative control were subjected to WGA. The WGA products and reference DNA were labeled with Cy3 and Cy5 fluorophores for 2 – 4 h. Labeled DNA

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was then resuspended in the hybridization buffer and hybridized onto the 24sure slides under cover slides for 4 – 6 h. After washing and drying, the slides were scanned at 10 μm

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using a laser scanner (Innoscan 710, Carbonne, France). The scanned data were then

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analyzed and quantified by algorithm fixed settings in BlueFuse Multi Software (BlueGnome, Cambridge, UK), a software package that performed the steps of grid placement,

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quantification, normalization and post-processing automatically. Once

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amplification was observed (i.e. low autosomal noise), autosomal profiles were analyzed for gain or loss of whole chromosomal ratios using a 3 × SD assessment, greater than ± 0.3 log2 ratio call, or both according to the manufacturer’s instructions. Of note, at our laboratory setting, the overall rate of mosaicism detected by aCGH for blastocyst biopsy was %5.3.

ACCEPTED MANUSCRIPT The vitrification and warming procedures, which we have been used in our laboratory setting, have been previously described by Cobo et al. (Cobo et al., 2008).

Single euploid blastocyst transfer was performed in a FER cycle in all patients in this arm.

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Artificial cycle was used for preparation of endometrium for FER cycles.

Data collection

Medical data on all couples with AMA included in the cohort were obtained from electronic

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records of the Anatolia Private IVF Centre and covered the period of June 2013 – June 2016. These data included female age, male age, female body mass index (BMI), duration of infertility, previous childbirth, the number of previous IVF cycles, information on the initial

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IVF cycle; number of oocytes retrieved and availability of an embryo to be transferred.

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Since the reasons for discontinuation were not recorded in medical files, a survey delineating the reason(s) for discontinuation was prepared. The current survey was inspired from

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previous reports (Verberg et al., 2008; Brandes et al., 2009). All the included patients were

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contacted by a phone call given by a single physician (Z.Y.C). Initially, the female partner was tried to be contacted; if this attempt was unsuccessful, then, the male partner was

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attempted to be contacted. At the phone call, the couple was asked why they discontinued their treatment. The reasons for discontinuation classified in the survey included advancing female age, psychological burden of the treatment, admitting to another IVF clinic, financial issue, time issue (logistic and practical reasons), no longer willing to undergo IVF treatment any more, spontaneous live birth, health problems, perception of poor prognosis and other reason(s) but not specified in the survey. We asked the couple for the leading reason for

ACCEPTED MANUSCRIPT their discontinuation of their further IVF attempts.

Statistical methods To compare the discontinuation rates between the two treatment strategies, Kaplan–Meier survival analysis was performed. For this analysis, discrete time variable was taken as the

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cycle number and censoring (drop-outs from the sample before the final outcome is observed) for patients who achieved an ongoing pregnancy with IVF during the study period.

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Cox’s proportional hazards analysis was performed to analyze the influence of potential independent risk factors on the risk of discontinuation both in the whole study population as well as in the PGT-A arm only.

Hazard ratio (HR) was used to calculate the risk of

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discontinuing from further IVF treatment for each potential factor.

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A chi-square test was performed to compare the frequency of leading reason given in the survey for discontinuation of the treatment protocols. A value of p <0.05 was considered to

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be statistically significant. Analyses were performed using SPSS version 21.0 (SPSS Inc.,

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Chicago, IL, USA, 1999).

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The Institutional Review Board of Hacettepe University approved the study protocol (KA180108).

Results The baseline demographic features of the patients, including female age, male age, antral follicle count, number of previous IVF attempts, number of patients with previous childbirth

ACCEPTED MANUSCRIPT were comparable between the PGT-A (198 patients; 285 cycles) and conventional IVF (203 patients; 270 cycles) arms (Table 1). Notably, the demographic features at the very first cycle were used in this analysis.

A consecutive 203 patients was included in the conventional IVF arm. Of these 203 patients,

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at the first attempt of OS, 144 had fresh embryo transfer with 25 ongoing pregnancies, whereas 59 had cycle cancellation before embryo transfer (Figure 1). The discontinuation rate following the 1st failed IVF attempt was 70.2% (125/178). Of the 53 patients starting the

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2nd cycle, 13 had cycle cancellation before embryo transfer, 26 had fresh embryo transfer, 14 had FER derived from the 1st OS resulting in a total of 7 (5 from second stimulation and 2 from first FER) ongoing pregnancies. Following second failed cycle, the discontinuation rate was 69.6% (32/46). Of the 14 patients starting the 3rd cycle, 1 had cycle cancellation, 9 had

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fresh embryo transfer, 4 had FER (1 from the 1st and 3 from the 2nd OS) resulting in only 1

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ongoing pregnancy from the fresh embryo transfer (Figure 1).

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A consecutive 198 patients were included in the PGT-A arm (Figure 2). Of these 198

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patients, at the first attempt of OS, 68 were cancelled at different stages up to at least one eligible blastocyst for biopsy, 69 had no eupolid embryo following biopsy, and the remaining

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61 had SFEET with 26 ongoing pregnancies (Figure 2). The discontinuation rate following the 1st failed PGT-A attempt was 62.8% (108/172). Of the 64 patients starting the 2nd cycle, 25 were cancelled at different stages up to at least one eligible blastocyst for biopsy, 13 had no eupolid embryo following biopsy, 15 had SFEET derived from 1st OS and 11 had SFEET derived from 2nd OS resulting in 10 ongoing pregnancies. Following second failed cycle, the discontinuation rate was 57.4% (31/54). Of the 23 patients starting the 3rd cycle, 8 were

ACCEPTED MANUSCRIPT cancelled at different stages up to at least one eligible blastocyst for biopsy, 7 had no eupolid embryo following biopsy, 1 had SFEET derived from 1st OS, 2 had SFEET derived from 2nd OS and the remaining 5 had SFEET derived from 3rd OS resulting in a total of 5 ongoing pregnancies (Figure 2). Of note, in the PGT-A arm, a total of 440 blastocysts were biopsied. Of these 440 blastocysts, 136 (30.9%) were euploid, 291 were aneuploid (66.1%), and the

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remaining 13 (3.0%) were mosaic.

The discontinuation rates following 1st failed IVF cycle were 62.8% vs. 70.2% in the PGT-A

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and conventional IVF arms, respectively (p=0.143). The respective figures for the 2nd cycle were 57.4% and 69.6% (p=0.209) (Table 2). The overall cumulative discontinuation rate for three planned cycles was 88.9% in the whole cohort; this figure was also comparable

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between the PGT-A and conventional IVF arms (85.8% and 91.8%, p=0.081, respectively, Table 2). The miscarriage and multiple pregnancy rates were less in the PGT-A arm when

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compared with the conventional IVF arm, but not reaching statistical significance (Table 2). The only multiple pregnancy in the PGT-A arm was a monozygotic twin. The cumulative

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ongoing pregnancy rate per patient was comparable between the two arms (20.7% vs.

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16.2%, p=0.369). However, ongoing pregnancy per embryo transfer was significantly higher

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in the PGT arm (42.2% vs. 16.4%, p<0.001).

Female age, male age, previous childbirth, antral follicle count, cycle cancellation in the first cycle, number of oocytes retrieved, performing PGT-A or not and availability of an embryo for transfer (untested in the conventional IVF arm and tested in the PGT-A arm) were assessed by the Cox-regression analysis (Table 3). Of these variables included, only the female age was noted to be the significant contributor to discontinue treatment (HR 1.07;

ACCEPTED MANUSCRIPT 95% CI, 1.09–1.13). Cox-regression analysis was performed in the PGT-A arm to delineate the effect of number of euploid blastocyst(s) on couples’ decision to discontinue treatment. When female age, male age, previous childbirth, antral follicle count, number of oocytes retrieved, cycle cancellation in the first cycle, and number of euploid blastocyst(s) were included as covariates, only the number of euploid blastocyst(s), but not the others,

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independently had an effect to discontinue treatment in PGT-A patients (HR 0.66; 95% CI, 0.50–0.87).

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Of the 296 couples discontinuing treatment in both arms, 179 (179/296; 60.5%) were reached by phone call (Figure 3). In the PGT-A arm, this figure was 81 (81/139; 58.3%), whereas, it was 98 (98/157; 62.4%) in the conventional IVF arm (p=0.46). Of the 117

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patients with no available survey result, 99 could not be reached by phone call and the remaining 18 did not wish to participate the survey despite being reached by phone call.

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The reasons for discontinuation of treatment are given in Figure 3. Overall, the most frequent reason for discontinuation of the treatment was psychological burden (37/179;

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20.7%). Although advancing female age (3.7% vs. 18.4%, p=0.002) and logistic/practical

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issues (3.7% vs. 15.3%, p= 0.011) were significantly less encountered as a reason to discontinue treatment in the PGT-A arm, continuing at another IVF clinic was a more

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frequent reason (21.0% vs. 10.2%, p= 0.045) when compared with the conventional IVF arm. The other reasons given in the survey included a previous healthy child (n=3) and unstable marital relationship (n=1) (Figure 3).

Discussion In our study, we noted that, overall, ~ 90% of AMA cases, not achieving an ongoing

ACCEPTED MANUSCRIPT pregnancy discontinued IVF treatment at a single center before completing three cycles. Performing PGT-A did neither reduce the discontinuation rate following the 1st, or 2nd failed cycle nor the cumulative rate before completing three cycles. Female ageing was the only independent factor to discontinue treatment with the hazard of 7% with each year of ageing. Overall, psychological burden was the main reason for treatment discontinuation as

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assessed by the survey.

There is a huge variation in the reported discontinuation rate for IVF treatment, ranging

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from 17-65% (Olivius et al., 2002; Sharma et al., 2002; Olivius et al., 2004; Soullier et al., 2008; Verberg et al., 2008; Troude et al., 2014). Variability may, in large part, be explained by the lack of consensus on i) definition of the final outcome which may influence number of

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censored cases (e.g. clinical pregnancy, ongoing pregnancy, live birth), ii) follow up period required to assign the event as discontinuation (Land et al., 1997; Pearson et al., 2009); (e.g.

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first failed IVF cycle or 6, 12 months following first failed IVF cycle) and iii) taking or not into account of patients who continue treatment at a different IVF clinic (Stolwijk et al., 1996;

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Verhagen et al., 2008). Other contributors may be IVF treatment coverage policy (Kulkarni et

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al., 2014), the type of population under study (e.g. poor prognosis patients) (Malizia et al.,

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2009; Troude et al., 2014) and OS protocols employed (Verberg et al., 2008).

Financial concern has been quoted to be one of the factors of discontinuation from subsequent IVF treatment (Dawson et al., 2005; McDowell and Murray 2011; Kulkarni et al., 2014). In the UK, 64% of couples discontinued treatment after one unsuccessful IVF attempt mainly due to financial concern (Sharma et al., 2002) as was also the case in some other studies (McDowell and Murray 2011; Kulkarni et al., 2014). However, financial constraint is

ACCEPTED MANUSCRIPT not the only reason as reflected by high discontinuation rates in countries where public funds cover IVF treatment (Olivius et al., 2002; Brandes et al., 2009; Troude et al., 2014). In a Dutch study, 57% of the couples discontinued IVF before three reimbursed cycles were completed (Brandes et al., 2009); in such circumstances, psychological burden was the main reason encountered in 65% of the discontinued cases (Rajkhowa et al., 2006; Brandes et al.,

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2009; Gameiro et al., 2012). Similarly, in our study, although IVF treatment was completely paid by patients on their own, psychological burden was the main reason for treatment

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discontinuation.

In a French study, Troude et al, by analyzing 5,135 couples undergoing IVF treatment at eight different centers, identified the following risk factors for early discontinuation following 1st

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or 2nd failed fresh/FER cycle: female age >34 years, duration of infertility >6 years, no or one oocyte retrieved and no embryo transfer at the first IVF cycle. Advancing female age was

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reported to be an independent factor for the risk of treatment discontinuation; the discontinuation rate was 22% in women aged 30–34 years, whereas, it was 35% in women

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≥40 years (Troude et al., 2014).

Our finding of advancing female age as the only

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independent risk factor for treatment discontinuation, with hazard of 7% with one year of

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ageing, is concordant with Troude, et al’s (2014) findings.

Aneuploidy associated with AMA is the main contributor for unsuccessful IVF attempts, increased risk of miscarriage and pregnancies with abnormal genotype (Hodes-Wertz et al., 2012; Franasiak et al., 2014). Since morphology is a poor predictor of embryo viability, and the prevalence of aneuploidy increases with AMA, PGT-A is commonly performed in AMA cases (Weissman et al., 2017). However, it must be noted that PGT-A does not improve per-

ACCEPTED MANUSCRIPT retrieval pregnancy rate, nor does it increase the cumulative ongoing/live birth rate, as was the case in our study; rather, it enables euploid embryos to be transferred and possibly decreases the risk of miscarriage. Since single euploid embryo is transferred in PGT-A cases, avoiding multiple pregnancies is another advantage of PGT-A compared with conventional IVF, with the very rare exception of mono-zygoting twinning cases as was the only multiple

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pregnancy in the PGT-A arm in our study. It should be also stressed that PGT-A has shortcomings, including a 1–5% misdiagnosis rate, a <1-2% embryo non-survival rate following thawing and lack of consensus on how to deal with mosaic embryos (Greco et al.,

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2015; Brezina et al., 2016; Scott and Galliano 2016). Moreover, the only available three randomized controlled trials (RCTs) on the efficacy of PGT-A are performed in good

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prognosis patients and there is an urgent need to perform RCTs in AMA cases.

In contrast to the theoretical expectation of less discontinuation rate in favor of PGT-A arm

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due to potential advantages of avoiding futile embryo transfer cycles and decreased risk of miscarriage, we did not notice any significant difference in the cumulative discontinuation

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rate before completing three cycles or following the 1st, or 2nd failed cycle in our study. On

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the other hand, as might be expected, the number of euploid blastocyst(s) independently decreased the discontinuation rate in PGT-A arm patients (HR 0.66; 95% CI, 0.50–0.87). Of

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interest, although the numbers were limited, financial issue as a reason for discontinuation was comparable between the two arms, as assessed by the survey. This finding is in contrast to the theoretical expectation that the additional expenses (trophectoderm biopsy, cryopreservation of embryos and genetic testing) in the PGT-A arm do not lead to higher discontinuation due to financial concerns.

Furthermore, significantly less frequency of

advancing female age as a reason of discontinuation in the PGT-A arm might be related with

ACCEPTED MANUSCRIPT a positive change of the couples’ attitude that may in turn increase the compliance for further attempts.

The main limitation of our study is its retrospective design. Although the baseline demographic features that may affect the ongoing pregnancy rate were comparable

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between the PGT-A and conventional IVF arms, the risk of unmeasured confounding factors cannot be ruled out. Although, the overall 60.5% contact rate by phone call to make the survey is not ideal, it was comparable between the PGT-A (58.3%) and conventional IVF In our survey, overall, psychological burden was the main reason for

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(62.4%) arms.

discontinuation and financial constraints were the fourth most prevalent reason. Due to limited sample size, it was not possible to delineate the impact of financial constraints, per

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se, on psychological burden. The use of 6-month as the time period of discontinuation is a limitation of the current series, although one may still consider 6-month as an acceptable

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time period for discontinuation in AMA cases at a single center. Taking ongoing pregnancy, rather than live birth, as treatment success is another limitation. Finally, considering those

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couples undergoing treatment at another IVF clinic, as treatment discontinuation at a single

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center, may not reflect real discontinuation from IVF treatment.

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We conclude that IVF treatment discontinuation in AMA cases is very frequent (~ 90%) but not influenced by performing PGT-A. Female ageing is the only significant contributor to discontinue treatment with the hazard of 7% with each year of ageing.

Overall,

psychological burden is the leading reason for the discontinuation of IVF treatment as assessed by survey.

ACCEPTED MANUSCRIPT REFERENCES Brandes, M., Van Der Steen, J.O., Bokdam, S.B., Hamilton, C.J., De Bruin, J.P., Nelen, W.L., Kremer, J.A., 2009. When and why do subfertile couples discontinue their fertility care? A longitudinal cohort study in a secondary care subfertility population. Hum. Reprod. 24, 3127-3135.

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Brezina, P.R., Kutteh, W.H., Bailey, A.P., Ke, R.W., 2016. Preimplantation genetic screening (pgs) is an excellent tool, but not perfect: A guide to counseling patients considering pgs. Fertil. Steril. 105, 49-50.

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Coates, A., Bankowski, B.J., Kung, A., Griffin, D.K., Munne, S., 2017. Differences in pregnancy outcomes in donor egg frozen embryo transfer (fet) cycles following preimplantation genetic screening (pgs): A single center retrospective study. J. Assist. Reprod. Genet.

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34, 71-78.

Cobo, A., Kuwayama, M., Perez, S., Ruiz, A., Pellicer, A., Remohi, J., 2008. Comparison of

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concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the cryotop method. Fertil. Steril. 89, 1657-1664.

PT

Dawson, A.A., Diedrich, K., Felberbaum, R.E., 2005. Why do couples refuse or discontinue

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art? Arch. Gynecol. Obstet. 273, 3-11. Franasiak, J.M., Forman, E.J., Hong, K.H., Werner, M.D., Upham, K.M., Treff, N.R., Scott, R.T.,

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Jr., 2014. The nature of aneuploidy with increasing age of the female partner: A review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening. Fertil. Steril. 101, 656-663 e651.

Gameiro, S., Boivin, J., Peronace, L., Verhaak, C.M., 2012. Why do patients discontinue fertility treatment? A systematic review of reasons and predictors of discontinuation in fertility treatment. Hum. Reprod. Update. 18, 652-669.

ACCEPTED MANUSCRIPT Greco, E., Minasi, M.G., Fiorentino, F., 2015. Healthy babies after intrauterine transfer of mosaic aneuploid blastocysts. N. Engl. J. Med. 373, 2089-2090. Hodes-Wertz, B., Grifo, J., Ghadir, S., Kaplan, B., Laskin, C.A., Glassner, M., Munne, S., 2012. Idiopathic recurrent miscarriage is caused mostly by aneuploid embryos. Fertil. Steril. 98, 675-680.

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Kulkarni, G., Mohanty, N.C., Mohanty, I.R., Jadhav, P., Boricha, B.G., 2014. Survey of reasons for discontinuation from in vitro fertilization treatment among couples attending infertility clinic. J. Hum. Reprod. Sci. 7, 249-254.

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Land, J.A., Courtar, D.A., Evers, J.L., 1997. Patient dropout in an assisted reproductive technology program: Implications for pregnancy rates. Fertil. Steril. 68, 278-281. Lande, Y., Seidman, D.S., Maman, E., Baum, M., Hourvitz, A., 2015. Why do couples

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discontinue unlimited free ivf treatments? Gynecol. Endocrinol. 31, 233-236. Malizia, B.A., Hacker, M.R., Penzias, A.S., 2009. Cumulative live-birth rates after in vitro

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fertilization. N. Engl. J. Med. 360, 236-243. Mcdowell, S., Murray, A., 2011. Barriers to continuing in vitro fertilisation--why do patients

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exit fertility treatment? Aust. N. Z. J. Obstet. Gynaecol. 51, 84-90.

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Mumusoglu, S., Yarali, I., Bozdag, G., Ozdemir, P., Polat, M., Sokmensuer, L.K., Yarali, H., 2017. Time-lapse morphokinetic assessment has low to moderate ability to predict

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euploidy when patient- and ovarian stimulation-related factors are taken into account with the use of clustered data analysis. Fertil. Steril. 107, 413-421 e414.

Olivius, C., Friden, B., Borg, G., Bergh, C., 2004. Why do couples discontinue in vitro fertilization treatment? A cohort study. Fertil. Steril. 81, 258-261.

ACCEPTED MANUSCRIPT Olivius, K., Friden, B., Lundin, K., Bergh, C., 2002. Cumulative probability of live birth after three in vitro fertilization/intracytoplasmic sperm injection cycles. Fertil. Steril. 77, 505-510. Pearson, K.R., Hauser, R., Cramer, D.W., Missmer, S.A., 2009. Point of failure as a predictor of in vitro fertilization treatment discontinuation. Fertil. Steril. 91, 1483-1485.

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Rajkhowa, M., Mcconnell, A., Thomas, G.E., 2006. Reasons for discontinuation of ivf treatment: A questionnaire study. Hum. Reprod. 21, 358-363.

Scott, R.T., Jr., Galliano, D., 2016. The challenge of embryonic mosaicism in preimplantation

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genetic screening. Fertil. Steril. 105, 1150-1152.

Sharma, V., Allgar, V., Rajkhowa, M., 2002. Factors influencing the cumulative conception rate and discontinuation of in vitro fertilization treatment for infertility. Fertil. Steril.

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78, 40-46.

Soullier, N., Bouyer, J., Pouly, J.L., Guibert, J., De La Rochebrochard, E., 2008. Estimating the

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success of an in vitro fertilization programme using multiple imputation. Hum Reprod. 23, 187-192.

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Stolwijk, A.M., Hamilton, C.J., Hollanders, J.M., Bastiaans, L.A., Zielhuis, G.A., 1996. A more

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realistic approach to the cumulative pregnancy rate after in-vitro fertilization. Hum. Reprod. 11, 660-663.

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Troude, P., Guibert, J., Bouyer, J., De La Rochebrochard, E., Group, D., 2014. Medical factors associated with early ivf discontinuation. Reprod. Biomed. Online. 28, 321-329.

Ubaldi, F.M., Capalbo, A., Colamaria, S., Ferrero, S., Maggiulli, R., Vajta, G., Sapienza, F., Cimadomo, D., Giuliani, M., Gravotta, E., Vaiarelli, A., Rienzi, L., 2015. Reduction of multiple pregnancies in the advanced maternal age population after implementation

ACCEPTED MANUSCRIPT of an elective single embryo transfer policy coupled with enhanced embryo selection: Pre- and post-intervention study. Hum. Reprod. 30, 2097-2106. Verberg, M.F., Eijkemans, M.J., Heijnen, E.M., Broekmans, F.J., De Klerk, C., Fauser, B.C., Macklon, N.S., 2008. Why do couples drop-out from ivf treatment? A prospective cohort study. Hum. Reprod. 23, 2050-2055.

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Verhagen, T.E., Dumoulin, J.C., Evers, J.L., Land, J.A., 2008. What is the most accurate estimate of pregnancy rates in ivf dropouts? Hum. Reprod. 23, 1793-1799.

Weissman, A., Shoham, G., Shoham, Z., Fishel, S., Leong, M., Yaron, Y., 2017. Preimplantation

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genetic screening: Results of a worldwide web-based survey. Reprod. Biomed.

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Online. 35, 693-700.

ACCEPTED MANUSCRIPT Authors’ roles S.M.: Conception of the research idea, design of the study, statistical analyses, drafting and reviewing the article for intellectual content. İ.Y.Ö.: Data collection, drafting the article. Z.Y.C.: Data collection, drafting the article. M.P.: Data collection, reviewing the article for intellectual content. L.K.S.: Reviewing the article for intellectual content. G.B.: Design of the

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study, drafting and reviewing the article for intellectual content. H.Y.: Conception of the research idea, design of the study, drafting and reviewing the article for intellectual content.

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Funding There was no funding.

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Conflict of Interest

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None declared.

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Author Biography

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Sezcan Mumusoglu is currently working as an assistant professor at the Department of OB/GYN, Hacettepe University. He completed his residency and clinical fellowship program

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in the Division of Reproductive Endocrinology and Infertility in the same university. His

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research interests are clinical IVF and polycystic ovary syndrome.

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Legends

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Figure 1. A flow chart of conventional IVF arm with ongoing pregnancies achieved and

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discontinuation rates

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rates

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Figure 2. A flow chart of PGT-A arm with ongoing pregnancies achieved and discontinuation

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Figure 3. Reasons for discontinuation in the PGT-A and conventional IVF arms as revealed by

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the survey (n= 179)

ACCEPTED MANUSCRIPT Table 1. Comparison of the baseline demographic features of the PGT-A (198 patients; 285 cycles) and conventional IVF (203 patients; 270 cycles) arms PGT-A arm

Conventional

P value

IVF arm

40.9 ± 2.2

40.7 ± 2.2

0.506

Male age, y

42.9 ± 6.2

42.6 ± 6.7

0.664

Female body mass index (kg/m2)

25.9 ± 5.3

26.4 ± 4.6

0.347

Duration of infertility (month)

56.7 ± 57.0

64.7 ± 57.7

0.206

Number of previous IVF cycles*

1.0 (0; 3)

1.0 (0; 3)

0.537

Antral follicle count

8.0 ± 5.3

7.7 ± 6.2

0.518

Previous childbirth, n (%)

49 (24.7)

43 (21.2)

0.233

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Female age, y

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Values are given as mean ± SD or median (25 th – 75 th percentiles), unless stated otherwise. *Not normally distributed

ACCEPTED MANUSCRIPT Table 2. Pregnancy outcome and discontinuation rates of the PGT-A (198 patients; 285 cycles) and conventional IVF (203 patients; 270 cycles) arms PGT-A arm

Conventional

P value

IVF arm Number of embryos transferred

1.0±0.0

1.76±0.48

<0.001

Clinical pregnancy rate per embryo

52/97 (53.6)

50/201 (24.9)

<0.001

Miscarriage rate, n (%)

11/52 (21.2)

17/50 (34.0)

0.146

Ongoing pregnancy rate per embryo

41/97 (42.2)

33/201 (16.4)

<0.001

41/198 (20.7)

33/203 (16.2)

0.369

Cumulative ongoing pregnancy rate per patient, n (%)

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transfer cycle, n (%)

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transfer, n (%)

Multiple pregnancy rate, n (%)

1/52 (1.9)

5/50 (10.0)

0.108

Discontinuation rate after first cycle, n

108/172 (62.8)

125/178 (70.2)

0.143

31/54 (57.4)

32/46 (69.6)

0.209

157/171 (91.8)

0.081

(%)

(%)

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completing three cycles

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Cumulative discontinuation rate before

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Discontinuation rate after second cycle, n

139/162 (85.8)

ACCEPTED MANUSCRIPT Table 3. Hazard ratios for discontinuing IVF treatment as computed by Cox regression analysis

Hazard ratio

P value

(95% CI) 1.071 (1.009 - 1.136)

0.025

Male age

0.990 (0.970 - 1.011)

0.354

Previous childbirth

0.910 (0.674 - 1.227)

0.535

Cycle cancelation

1.289 (0.900 - 1.846)

0.165

Antral follicle count

1.001 (0.973 - 1.030)

0.952

Number of oocytes retrieved

0.985 (0.941 - 1.030)

0.504

IVF treatment policy (PGT-A; conventional IVF) Availability of an embryo for transfer * *

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Female age

0.817 (0.547 - 1.221)

0.324

0.963 (0.602 - 1.541)

0.875

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Tested in the PGT-A arm, untested in the conventional IVF arm