pregnancy loss patients with diminished ovarian reserve

(1)

Higher rates of aneuploidy in

blastocysts and higher risk of no embryo transfer in recurrent

pregnancy loss patients with diminished ovarian reserve

undergoing in vitro fertilization

Lora K. Shahine, M.D., Lorna Marshall, M.D., Julie D. Lamb, M.D., and Lee R. Hickok, M.D.

Paciﬁc NW Fertility, Seattle, Washington

Objective: To study the prediction of aneuploidy rate in blastocysts from patients with recurrent pregnancy loss (RPL) on the basis of ovarian reserve testing.

Design: Prospective cohort analysis.

Setting: Private, university-afﬁliated fertility clinic.

Patient(s): A total of 239 patients with RPL, deﬁned as two or more clinical miscarriages, were screened for inclusion. One hundred two (102) cycles in patients with unexplained RPL resulted in at least one euploid embryo transferred. Outcomes were compared by ovarian reserve test results, with diminished ovarian reserve (DOR) deﬁned as a cycle day 3 FSH >10 IU/mL and/or antim€ullerian hormone

<1 ng/mL.

Intervention(s): In vitro fertilization with blastocyst biopsy and aneuploidy screening of all 23 chromosome pairs.

Main Outcome Measure(s): Rate of aneuploidy in blastocysts and incidence of IVF cycles with no transfer owing to no euploid blasts.

Result(s): Patients with DOR had a higher percentage of aneuploid blastocysts (57% vs 49%) and a higher incidence of no euploid embryos to transfer (25% vs 13%). The higher rate of aneuploidy in blastocysts was most signiﬁcant in patients aged <38 years (67% vs 53%). Im- plantation rates after transfer of euploid blastocysts were similar (61% compared with 59%), and miscarriage rates were low (14% and 10%).

Conclusion(s): Unexplained RPL patients with DOR have a higher percentage of aneuploid blastocysts and risk of no euploid embryo to transfer compared with unexplained RPL patients with normal ovarian reserve testing. The difference is most signiﬁcant in patients aged <38 years. Patients with RPL and DOR with euploid embryo transferred had similar outcomes compared with patients with RPL and normal ovarian reserve testing. (Fertil Steril^Ò2016;106:1124–8. Ó2016 by American Society for Reproductive Medicine.) Key Words: Aneuploidy, diminished ovarian reserve, in vitro fertilization, preimplantation genetic screening, recurrent pregnancy loss Discuss: You can discuss this article with its authors and with other ASRM members athttps://www.fertstertdialog.com/users/

16110-fertility-and-sterility/posts/10951-higher-rates-of-aneuploidy-in-blastocysts-and-higher-risk-of-no-embryo-transfer- in-recurrent-pregnancy-loss-patients-with-diminished-ovarian-reserve-undergoing-in-vitro-fertilization

R

ecurrent pregnancy loss (RPL) is a clinically frustrating and emotionally charged challenge

for patients and providers alike. In 2013 the American Society of Repro- ductive Medicine (ASRM) stated that

RPL is a disease distinct from infertility, which is deﬁned by two or more clinically recognized pregnancy losses (1). An evaluation of a couple with RPL, which includes parental karyo- types, uterine cavity evaluation, hor- monal testing, and screening for antiphospholipid syndrome, will ﬁnd a cause for RPL in less than 50% of cases(2, 3).

A high incidence of aneuploidy in products of conception from

Received February 3, 2016; revised and accepted June 7, 2016; published online June 29, 2016.

L.K.S. has nothing to disclose. L.M. has nothing to disclose. J.D.L. has nothing to disclose. L.R.H. has nothing to disclose.

Presented as an oral presentation at the Paciﬁc Coast Reproductive Society Meeting, Turtle Bay, HI, September 1, 2015.

Reprint requests: Lora K. Shahine, M.D., Paciﬁc NW Fertility, 1101 Madison Street, Ste. 1050, Seattle, Washington 98104 (E-mail:lshahine@pnwfertility.com).

Fertility and Sterility® Vol. 106, No. 5, October 2016 0015-0282/$36.00

http://dx.doi.org/10.1016/j.fertnstert.2016.06.016

(2)

ﬁrst-trimester losses can potentially explain repeated miscarriage in patients with otherwise unexplained RPL(2, 4, 5). The incidence of aneuploidy increases with maternal age(6)and number of previous miscarriages (7). In vitro fertilization with chromosomal screening (CS) of embryos for aneuploidy has been proposed as a treatment option for women with RPL(8).

Katz-Jaffe et al.(9)showed a higher incidence of aneuploid blasts in patients with diminished ovarian reserve (DOR), and other studies have shown a higher than expected incidence of DOR in RPL patients (10, 11). No published studies have shown what patients with RPL and DOR, compared with other patients with unexplained RPL, might expect should they undergo an IVF cycle.

The objective of this study was to examine the role of ovarian reserve testing in predicting rate of aneuploidy and the risk of having no euploid embryo to transfer in patients with unexplained RPL who choose IVF with CS as a treatment option.

MATERIALS AND METHODS

This study is a prospective cohort study of IVF outcomes in unexplained RPL patients who underwent IVF with preimplantation CS from January 1, 2011 to March 31, 2015 at a private, university-afﬁliated fertility center in Seattle, Wash- ington. Institutional review board approval was obtained for the project.

Clinical miscarriage was defined as pregnancy loss before 20 weeks' gestation after confirmation with ultrasound (gestational sac or fetal pole) or products of conception diag- nosed histologically by patient report or medical records. Pa- tients presenting for RPL were offered an evaluation if they had a history of two or more clinical miscarriages. Unex- plained RPL was defined as patients without uterine cavity defects, balanced translocation, antiphospholipid syndrome, or endocrine disorders(12).

Patients underwent ovarian reserve testing with both serum antim€ullerian hormone (AMH) levels and cycle day 2 or 3 serum FSH and E2, although this is not included in ASRM guidelines for evaluation of RPL (12). Follicle-stimulating hormone was measured by electrochemiluminescent immunoassay, and AMH was measured with an ELISA (Gen II ELISA reference A79765) (13, 14). A patient was considered to have DOR if their FSH was R10 mIU/mL and/or their AMH level was <1 ng/mL(13, 15). Antral follicle count was not used as a measure of ovarian reserve owing to its subjective nature and the previous description of the close correlation of antral follicle count and serum AMH(15). Patients were excluded if they had uncorrectable uterine cavity defects, antiphospholipid syndrome, or balanced translocations.

Standard protocols for controlled ovarian hyperstimulation were used. Protocol and dose of medication was deter- mined for individual patients by their provider, with the decision based on patient's age, history, and ovarian reserve testing. Protocols for normal ovarian reserve patients included either the use of a mid-luteal phase GnRH agonist for 7 days before initiation of gonadotropin stimulation or a GnRH antagonist initiated after gonadotropin start when

the lead follicle reached 12–14 mm, using an average daily gonadotropin dose of 300 IU. Protocols for poor ovarian reserve patients included either an antagonist protocol or microdose ﬂare protocol with a maximum daily gonadotropin dose of 450 IU. Human chorionic gonadotropin (10,000 IU) was administered when two lead follicles reached a mean diameter of 18–20 mm. A dual trigger of leuprolide acetate with 1,500 IU of hCG was used if patients were considered at risk for ovarian hyperstimulation syndrome (E₂ level

>4,000 pg/mL, 20þ mature follicles, or were symptomatic).

The transvaginal ultrasound-guided oocyte aspiration was performed 35 hours after the patient received the trigger shot.

In vitro fertilization, embryo culture, and blastocyst biopsy techniques were performed in a manner previously described by others (16). Intracytoplasmic sperm injection was performed in all cases. Trophectoderm biopsy was performed on all expanding or fully expanded blastocysts on postretrieval day 5 or 6, depending on the rate at which individual embryos reached this developmental stage. All embryos underwent vitriﬁcation after trophectoderm biopsy.

Each trophectoderm biopsy sample underwent CS analysis at a commercial preimplantation genetic diagnosis laboratory (Genesis Genetics, false-positive and false-negative rates of 2%, respectively). Metaphase comparative genomic hybridization with microarray analysis was used.

Only euploid embryos were warmed and subsequently transferred after adequate endometrial preparation and luteal support. Frozen embryo transfer protocols included either a programmed cycle with oral contraceptive pill preparation and estrogen supplement in the form of E2patches or IM estrogen valerate, or a natural protocol in which patients were monitored through their own menstrual cycle. Luteal support in the form of IM P or vaginal suppository was begun after the endometrial lining reached 8 mm and a serum E₂level of R150 pg/mL was observed. Embryo transfer occurred on the sixth day of P supplementation in a programmed cycle and on the sixth day after hCG administration in a natural cycle.

All patients had a normal uterine cavity evaluation before transfer. Embryo transfer was performed under transabdomi- nal ultrasound guidance after embryo survival was confirmed after warming. Either one or two embryos were transferred, with the majority of patients (93%) having a single embryo transferred. A serum pregnancy test was obtained 10 days after ET, and a transvaginal ultrasound was performed at approxi- mately 6 and 8 weeks' gestation. Implantation rate was defined as the number of gestational sacs noted on ultrasound exam- ination per number of embryos transferred. Data were analyzed with Student t tests and c²tests as appropriate, and a P value < .05 was considered to be statistically significant.

RESULTS

All patients presenting to our clinic from January 1, 2011 to March 31, 2015 were screened for inclusion. Patients with unexplained RPL who opted for IVF with preimplantation CS as a treatment option and had embryos screened for aneuploidy were included in the analysis. A total of 239 patients had two or more clinical miscarriages and received the ASRM evaluation for RPL(12). Of these, 179 patients had no explanation

(3)

for their RPL, and 60 patients had either explained RPL (20 patients had antiphospholipid antibodies, 10 had a uterine factor, 5 patients had a balanced translocation) or had incom- plete testing (25 total patients). The patients with unexplained RPL were offered expectant management with supportive care or IVF with CS of their embryos. Of the 179 unexplained RPL patients, 119 opted for IVF as a treatment, and 111 patients made it to retrieval. Eight IVF cycles were canceled for poor response, deﬁned as fewer than four mature follicles at time of hCG trigger, and all patients canceled had DOR. Of the 111 retrieval cycles, 102 cycles had blasts to screen for aneuploidy (5 cycles had no blast formation, and four patients declined CS after retrieval and opted for a fresh embryo transfer).

Patients who met inclusion criteria for analysis (unexplained RPL patients who opted for IVF with CS who had blastocysts available for biopsy) were analyzed by ovarian reserve test results. Fifty-nine patients had normal ovarian reserve testing, and 43 had DOR. Baseline characteristics of the patients are displayed in Table 1. The patient groups did not differ in average age, body mass index, average number of previous miscarriages, or number of patients with a history of a documented aneuploid miscarriage.

In vitro fertilization cycle characteristics did not differ for the most part between the groups (Table 2). Of the three stimulation protocols used, 78% of cycles were GnRH antagonist protocol, 17% were GnRH agonist, and 5% were microdose ﬂare protocol with GnRH agonist. There were more GnRH agonist cycles in the normal ovarian reserve group compared with the DOR group (12% vs 5% overall), and microdose ﬂare protocol was only used in DOR patients, but outcomes did not differ according to stimulation protocol. The majority of frozen embryo transfer cycles were programmed or medicated cycles (86%) compared with the natural cycles (14%). Howev- er, the rates of use were similar between the study groups.

Patients with normal ovarian reserve had a lower total amount of gonadotropins, a higher average number of mature eggs retrieved, and a higher average number of blastocysts available for biopsy and CS. Patients with DOR had a higher percentage of aneuploid blastocysts (57% vs 48%, P¼ .03).

The DOR group also had a higher incidence of IVF cycles in which all blastocysts tested were aneuploid, so no euploid embryos were available for transfer (25% vs 13%, P¼ .02).

Aneuploidy rate in embryos increased with advancing age in all unexplained RPL patients. However, the rate of aneuploidy differed according to ovarian reserve testing in similar age groups (Table 3). This difference is most signiﬁ- cant in patients aged <38 years. At age 35 years or less and ages 35–37 years, a higher rate of aneuploidy was found in embryos from patients with DOR compared with patients with normal ovarian reserve testing (59% vs 45%, P¼ .04 and 77% vs 59%, P¼ .03, respectively). In all patients aged

<38 years, a higher rate of aneuploidy was seen in embryos from DOR patients compared with patients with normal ovarian reserve testing (67% vs 53%, P¼ .04). In patients aged R38 years the aneuploidy rates were similarly high, despite differences in ovarian reserve testing (Table 3).

Of the 119 patients who chose IVF with CS, 102 had a blastocyst tested (85%) and 83 had a euploid embryo transferred (69%) (Table 4). Pregnancy outcomes did not differ in the two groups for patients who had a euploid embryo to transfer, despite the difference in ovarian reserve testing between the two groups. Implantation rates were similar between the normal ovarian reserve group and the DOR group (61% vs 59%, P¼ .3). Live birth rates were similar (54% vs 47%, P¼ .6). Miscarriage rates were slightly higher in the DOR group, but it was not a statistically signiﬁcant difference (14% vs 10%, P¼ .08). All patients with euploid embryos were counseled to have a single embryo transfer, and the average number of embryos transferred was 1.1 in the normal ovarian reserve group and 1.3 in the DOR group (93% of patients had a single euploid embryo transferred).

TABLE 1

Patient characteristics.

Characteristic

Normal ovarian

reserve (n [ 59) DOR (n [ 43) P value

Age (y) 36.1 (29–41) 37.0 (32–41) .09

BMI (kg/m²) 22.8 (18.7–27.5) 23.1 (19.4–29.1) .6 FSH (mIU/mL) 7.4 (4.5–9.7) 10.8 (6.4–16.1) .01 AMH (ng/mL) 2.9 (1.0–7.7) 1.3 (0.4–5.1) <.01 No. of clinical

miscarriages 2.5 (2–5) 2.6 (2–4) .7

History of aneuploid loss (%)

17 20 .1

Note: Values are averages unless otherwise noted.

Shahine. High aneuploidy in RPL patients with DOR. Fertil Steril 2016.

TABLE 2

Cycle characteristics Characteristic

Normal ovarian

reserve (n [ 59) DOR (n [ 43) P value Total amount of

gonadotropins (IU)

3,204.5 1,124.5 4,432.5 1,562.4 .04 No. of mature

eggs

12.9 (8–33) 8.5 (5–10) .01 No. of blasts

biopsied

5.3 (1–10) 3.6 (2–6) .02

Aneuploid blasts (%)

48 57 .03

% All aneuploid blasts

13 25 .02

Note: Values are averages unless otherwise noted.

TABLE 3

Percentage of aneuploid embryos according to age.

Age (y)

Normal ovarian reserve (n [ 59)

DOR

(n [ 43) P value

<35 45 59 .04

35–37 59 77 .03

38–40 74 76 .9

41þ 87 92 .8

Note: Values are percentages.

(4)

DISCUSSION

To our knowledge this is the ﬁrst study to show a higher rate of aneuploid embryos in patients with unexplained RPL and DOR compared with patients with unexplained RPL and normal ovarian reserve testing. This ﬁnding was most significant in patients aged <38 years. Interestingly, in patients who had euploid embryo(s) to transfer, the implantation potential after transfer was similar between the two groups, and miscarriage rates were low in both groups, despite differences in ovarian reserve testing. This study highlights two topics of interest: IVF with CS as a treatment option for RPL patients, and ovarian reserve testing for RPL patients.

With the high incidence of aneuploidy in products of conception from ﬁrst-trimester miscarriages (2, 4, 5), IVF with CS of embryos has been proposed as a treatment option for RPL patients with no identiﬁable explanation for their miscarriages (8). The logic is clear: unexplained RPL may be recurrent aneuploid pregnancies, and the technology to screen for euploid embryos before implantation should decrease the risk of miscarriage. There are several studies in the literature reporting on IVF with CS as a treatment option for unexplained RPL (17–22). The majority of these studies report on success rates with euploid transfer and compare miscarriage rate of patients with euploid embryo transferred with their age-related risk of miscarriage. It is encouraging that patients who have a euploid embryo have a high success rate with transfer and a lower than expected miscarriage rate for their age, but that is only part of the big picture. These types of studies do not highlight the patients who want to do IVF and CS for treatment but do not have an ET owing either to poor response or no euploid embryos to transfer. Counseling RPL patients about IVF with CS as a treatment option should include overall chances of success (not just the success rate if they have a euploid embryo transferred). This article suggests that women with unexplained RPL and DOR should be counseled to have different expectations as they enter an IVF cycle than similar patients with normal ovarian reserve.

Ovarian reserve testing is currently not considered a part of the RPL evaluation according to the Practice Committee Opinion for ASRM from 2012(12). One can argue that ovarian reserve testing is most useful for predicting response to ovarian stimulation and may not predict successful pregnancy when trying to conceive naturally. Our study found a

difference in incidence of aneuploid embryos based on ovarian reserve testing in unexplained RPL patients, especially in women aged <38 years. Ovarian reserve testing results can be used by clinicians to counsel patients considering IVF with CS as a treatment option for unexplained RPL. Given the high cost of IVF, patients with unexplained RPL and DOR, especially those who are easily conceiving spontaneously, should have realistic expectations and be counseled about all viable options, including expectant management and donor egg. Some studies have shown a higher rate of DOR in RPL patients(10, 11), and it may be reasonable to consider ovarian reserve testing early in the RPL evaluation when patients express an interest in IVF with CS as a potential treatment option.

Diminished ovarian reserve has been associated with higher rates of aneuploidy (9). However, other factors may contribute to aneuploidy rate, such as advanced age and gonadotropin dose. Prevalence of aneuploidy in embryos and miscarriages increases with age (6, 8), and our study found similarly high aneuploidy rates in patients aged

>38 years despite ovarian reserve testing results.

Interestingly, rates of aneuploidy were higher in patients with unexplained RPL with DOR compared with unexplained RPL patients with normal ovarian reserve testing at younger ages. The association of DOR and aneuploidy in unexplained RPL patients seems to be more significant in younger patients, and this finding may help in counseling younger patients with RPL on success rates with treatment options including IVF with CS. In addition, patients with DOR received a higher dose of gonadotropins than patients with normal ovarian reserve testing in our study. Some studies suggest that higher gonadotropins may be associated with aneuploidy (23), whereas other studies do not find this association (24, 25). Aneuploidy is multifactorial, and contributing factors to increased incidence of aneuploidy in IVF treatment remain under investigation.

The ASRM guidelines for evaluation and treatment of RPL do not currently suggest IVF with CS as a treatment option for unexplained RPL and note that it is important to emphasize to patients with unexplained RPL that the chance for a future successful pregnancy can exceed 50%–60%, depending on maternal age and parity (12). Patients and clinicians alike can ﬁnd it challenging to ﬁnd no cause for RPL and then recommend trying again to conceive without intervention.

Therefore, IVF with CS has been proposed to decrease risk of miscarriage from aneuploidy. One may argue that unexplained RPL patients may not need IVF with CS as an intervention, given the high cost of treatment. However, patients and clinicians alike are drawn to the idea of decreasing risk of miscarriage from aneuploidy with screening embryos.

The results from this study should help clinicians counsel patients with RPL on the overall chances of success with IVF based on ovarian reserve testing.

Similar to previous studies, we found a high success rate and low miscarriage rate in unexplained RPL patients opting for IVF with CS as a treatment option who had a euploid embryo to transfer. However, we found a signiﬁcantly higher rate of aneuploid embryos and cycles with no euploid embryos to transfer in unexplained RPL patients with DOR TABLE 4

Outcomes for cycles with euploid blast transferred.

Outcome

Normal ovarian reserve (n [ 51)

DOR

(n [ 32) P value

Euploid embryos transferred 1.1 1.3 .61

Implantation rate^aof euploid blasts transferred (%)

61 59 .45

Clinical miscarriage per transfer (%)

10 14 .08

Live birth per transfer (%) 54 47 .52

aNumber of gestational sacs/embryos transferred.

(5)

compared with unexplained RPL patients with normal ovarian reserve testing, especially in women aged

<38 years. To our knowledge this is the ﬁrst report to show this association of ovarian reserve testing and aneuploidy rates in patients with unexplained RPL. Our ﬁndings should help clinicians counsel patients about expectations and success when considering IVF with CS as a treatment option for these patients. Ovarian reserve testing should be considered early in the evaluation if patients are considering IVF with CS treatment for unexplained RPL, and patients with DOR should be well counseled about the overall success rate with all treatment options.

REFERENCES

1. Practice Committee of American Society of Reproductive Medicine. Deﬁni- tions of infertility and recurrent pregnancy loss: a committee opinion. Fertil Steril 2013;99:63.

2. Stephenson MD. Frequency of factors associated with habitual abortion in 197 couples. Fertil Steril 1996;66:24–9.

3. Jaslow CR, Carney JL, Kutteh WH. Diagnostic factors identiﬁed in 1020 women with two versus three or more recurrent pregnancy losses. Fertil Steril 2010;93:1234–43.

4. Daniely M, Aviram-Goldring A, Barkai G, Goldman B. Detection of chromosomal aberration in fetuses arising from recurrent spontaneous abortion by comparative genomic hybridization. Hum Reprod 1998;13:805–9. 5. Carp H, Toder V, Aviram A, Daniely M, Mashiach S, Barkai G. Karyotype of

the abortus in recurrent miscarriage. Fertil Steril 2001;75:678–82. 6. Marquard K, Westphal L, Milki A, Lathi R. Etiology of recurrent pregnancy

loss in women over the age of 35 years. Fertil Steril 2010;94:1473–7. 7. Kiss A, Rosa RF, Dibi RP, Zen PR, Pfeil JN, Graziadio C, et al. Chromosomal

abnormalities in couples with history of recurrent abortion. Rev Bras Ginecol Obstet 2009;31:68–74.

8. Munne S, Chen S, Fischer J, Colls P, Zheng X, Stevens J, et al. Preimplanta- tion genetic diagnosis reduces pregnancy loss in women aged 35 years and older with a history of recurrent miscarriages. Fertil Steril 2005;84:331–5. 9. Katz-Jaffe MG, Surrey ES, Minjarez DA, Gustofson RL, Stevens JM,

Schoolcraft WB. Association of abnormal ovarian reserve parameters with a higher incidence of aneuploidy blastocysts. Obstet Gynecol 2013;121:

71–7.

10. Trout SW, Seifer DB. Do women with unexplained recurrent pregnancy loss have higher day 3 serum FSH and estradiol values? Fertil Steril 2000;74:335–7.

11. G€urb€uz B, Yalti S, Ozden S, Ficicioglu C. High basal estradiol level and FSH/LH ratio in unexplained recurrent pregnancy loss. Arch Gynecol Obstet 2004;

270:37–9.

12. Practice Committee Opinion of the American Society for Reproductive Med- icine. Evaluation and treatment of recurrent pregnancy loss: a committee opinion. Fertil Steril 2012;98:1103–11.

13. Broekmans F, Kwee J, Hendriks D, Mol B, Lambalk C. A systematic review of tests predicting ovarian reserve and IVF outcome. Hum Reprod Update 2006;12:685–718.

14. Han X, McShane M, Sahertian R, White C, Ledger W. Pre-mixing serum sam- ples with assay buffer is a prerequisite for reproducible anti-Mullerian hormone measurement using the Beckman Coulter Gen II assay. Hum Reprod 2014;29:1042–8.

15. Broer S, Mol B, Hendriks D, Broekmans F. The role of anti-Mullerian hormone in prediction of outcome after IVF: comparison with the antral follicle count. Fertil Steril 2009;91:705–14.

16. Schoolcraft W, Fragouli E, Stevens J, Munne S, Katz-Jaffe M, Wells D. Clinical application of comprehensive chromosomal screening at the blastocyst stage. Fertil Steril 2010;94:1700–6.

17. Hill DL, Buehler Z, Jiang Z, Surrey M, Danzer H, Li M. P-27: in vitro fertilization – embryo transfer combined with aneuploidy screening of day three embryos lowers the miscarriage rate in recurrent pregnancy loss patients of advanced maternal age. Fertil Steril 2006;85 Suppl 2:21.

18. Garrisi J, Cohen J, Ferry KM, Colls P, Garrisi MG, Munne S. Preimplantation genetic diagnosis (PGD) effectively reduces idiopathic recurrent pregnancy loss (RPL) among patients with up to ﬁve previous miscarriages after natural conception. Fertil Steril 2007;88:S85.

19. Munne S, Fischer J, N-neka G, Escudero T, Zheng X, Colls P. Preimplan- tation genetic screening for recurrent pregnancy loss. Fertil Steril 2008;

90:S24.

20. Griﬁo J, Ghadir S, Kaplan B, Laskin C, Glassner M, Munne S. Signiﬁcant decrease in miscarriages after preimplantation genetic diagnosis (PGD) for recurrent pregnancy loss using array comparative genome hybridization (Array CGH). Fertil Steril 2011;96:S23.

21. Hodes-Wertz B, Grifo J, Ghadir S, Kaplan B, Laskin CA, Glassner M, et al.

Idiopathic recurrent miscarriage is caused mostly by aneuploid embryos. Fer- til Steril 2012;98:675–80.

22. Franasiak JM, Barnett R, Hong KH, Werner MD, Scott RT. Proportion of aneuploidy does not impact live birth rate or pregnancy loss rate in patients with recurrent pregnancy loss (RPL) undergoing comprehensive chromosome screening. Fertil Steril 2014;102:e171–2.

23. Braga DP, Setti AS, Figueira Rde C, Iaconelli A, Borges E. Contributing factors for the incidence of aneuploidy in older patients undergoing intracytoplasmic sperm injection cycles. J Assist Reprod Genet 2012;29:911–6. 24. Baart EB, Martini E, Eijkemans MJ, Van Opstal D, Beckers NG, Verhoeff A,

et al. Milder ovarian stimulation for in-vitro fertilization reduces aneuploidy in the human preimplantation embryo: a randomized controlled trial. Hum Reprod 2007;22:980–8.

25. Massie JA, Shahine LK, Milki AA, Westphal LM, Lathi RB. Ovarian stimulation and the risk of aneuploid conceptions. Fertil Steril 2011;95:970–2.