Preimplantation genetic diagnosis (PGD) improves pregnancy outcome for translocation carriers with a history of recurrent losses

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1 RECURRENT PREGNANCY LOSS Preimplantation genetic diagnosis (PGD) improves pregnancy outcome for translocation carriers with a history of recurrent losses Jill Fischer, M.S., Pere Colls, Ph.D., Tomas Escudero, M.S., and Santiago Munne, Ph.D. Reprogenetics, LLC, Livingston, New Jersey Objective: To determine if preimplantation genetic diagnosis (PGD) for translocation carriers with three or more pregnancy losses reduces loss rates. Design: Retrospective review of data. Setting: Preimplantation genetic diagnosis laboratory servicing IVF groups. Patient(s): Patients (n ¼ 192) undergoing PGD for either a reciprocal translocation or Robertsonian translocation who had three or more previous pregnancy losses. Intervention(s): Preimplantation genetic diagnosis for translocations. Main Outcome Measure(s): Pregnancy loss rate, pregnancy success rate defined as delivery of at least one child or an ongoing pregnancy in the third trimester, and length of time to success. Result(s): Pregnancy loss rate was significantly reduced to 13% post-pgd compared with 88.5% in previous non- PGD pregnancies and to 35% to 64% from naturally conceived pregnancies as reported in the literature. Pregnancy success rate was 87%. Conception occurred after an average of 1.4 cycles or <4 months. Conclusion(s): Individuals with translocations who have experienced three or more losses benefit from PGD by realizing a significant reduction in loss rate and improvement in rate of success of pregnancy. Length of time to conceive is also dramatically reduced compared with data in the literature for similar populations not undergoing PGD. (Fertil Steril Ò 2010;94: Ó2010 by American Society for Reproductive Medicine.) Key Words: Preimplantation genetic diagnosis, reciprocal translocation, Robertsonian translocation, recurrent pregnancy loss, miscarriage Individuals with translocations are known to have high rates of unbalanced gametes (1 6), have impaired or reduced gametogenesis (2, 6 10), produce high rates of unbalanced embryos (11, 12), and are therefore at risk for infertility and pregnancy loss (13). When conceiving naturally, these individuals experience loss in most pregnancies (11, 12). The prevailing attitude among various medical specialties is that, because most of the unbalanced pregnancies will miscarry and seldom reach term (14), further interventions other than idiopathic recurrent pregnancy loss (RPL) treatments are unnecessary. However, this attitude does not take into account the pain and suffering caused by RPL. Although the ultimate goal of translocation carriers is to achieve a viable pregnancy free of chromosome abnormalities, reducing the risk of miscarriage is a parallel goal. Previous studies have shown that preimplantation genetic diagnosis (PGD) for translocations reduced loss Received August 1, 2008; revised February 4, 2009; accepted February 19, 2009; published online December 24, J.F. has nothing to disclose. P.C. has nothing to disclose. T.E. has nothing to disclose. S.M. has nothing to disclose. Reprint requests: Jill Fischer, M.S., Reprogenetics, LLC, 3 Regent Street, Suite 301, Livingston, NJ (FAX: ; jfischer@ reprogenetics.com). rates from >90% to <15% (11, 12, 15 19). Additionally, studies of PGD for patients with idiopathic RPL, typically defined as three or more losses, have shown similar rates of reduction (20, 21). However, few PGD studies have looked at the subpopulation of RPL that are carriers of translocations who have had three or more losses. Recently, Otani et al. (19) undertook such a study and found a substantial reduction in miscarriages. Other studies have focused on natural conception of carriers of structural chromosome aberrations (14, 22, 23) either because of lack of availability of PGD or preference to not prescribe such as an option to conception. Lack of availability of PGD is understandable because of country of origin and governmental restrictions thereof. However, promotion of conceiving naturally despite the availability of PGD may overlook the patient s emotional state (19). We report on the largest dataset of PGD for chromosomal structural aberrations in Fischer et al. (in preparation). From that dataset we show here the outcome data on a subset of PGD for translocation patients who had three or more losses in previous non-pgd pregnancies. Instead of using the typical assisted reproductive technology (ART) definition of a successful pregnancy as a sac seen /$36.00 Fertility and Sterility â Vol. 94, No. 1, June doi: /j.fertnstert Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 after an IVF or other ART cycle, we use the definition of success typical to the RPL literature stated as survival past 24 weeks gestation. This definition is more useful for this article, as we are comparing outcome to the RPL data in the literature. Additionally, comparison would be difficult with the ART definition of success as it relies on pregnancy per attempt, and one cannot assess how many times the non-art population attempts to conceive. MATERIALS AND METHODS Patients Through March 2008, PGD for structural chromosome aberrations was performed in 1,060 cycles, the largest dataset reported to date. Samples were sent from 116 IVF centers to our lab for analysis. Testing was performed for couples in which one or both members had a chromosomal structural aberration such as a translocation. A total of 683 patients underwent the 1,060 cycles. Previous pregnancy information was known for 807 of the 1,060 cycles (76%). Of these, 192 patients undergoing PGD for either a reciprocal translocation or Robertsonian translocation had three or more previous pregnancy losses. These 192 patients were selected as the population of interest for this article. For these RPL patients, the average maternal age was 34 and the average number of previous losses was 3.8 (range: 3 7). Preimplantation Genetic Diagnosis Previous to IVF and PGD, patients underwent evaluations used for IVF and RPL patients such as history and physical exam, saline sonogram, hysterosalpingogram, TSH, prolactin, thyroid peroxidase antibodies, antithyroglobulin antibodies, lupus anticoagulant, anticardiolipin antibodies, antiphosphatidyl serine, beta 2 glycoprotein antibodies, fasting blood sugar, complete metabolic panel, complete blood count, hemoglobin electrophoresis, indirect Coombs, homocysteine, lupus anticoagulant, anticardiolipin, antiphosphatidyl serine, antithrombin III, Protein C, Protein S total and free, factor V and II mutation analysis, and beta 2 glycoprotein antibody testing. Chromosome analyses were performed on the spouse if not previously done. Preimplantation genetic diagnosis via fluorescence in situ hybridization (FISH) was performed on either polar bodies or blastomeres obtained from day 3 embryos. Embryo transfer occurred on day 4 or 5 of development as determined by the timing of the reporting of the PGD results or per preference of the physician at the IVF group. The vast majority of cycles were performed by embryo biopsy. For embryo biopsy cycles, one cell per embryo at day 3 was biopsied and fixed on a glass slide using the modified Carnoy method as previously described (24). The single cell analysis was conducted following strict assessment criteria described by Munne (25). For PB biopsy cases, the same procedure described in Munne et al. (11) was followed without modification. For reciprocal translocation cases, preliminary testing of probes was completed in blood of the carriers of the aberration to confirm the chosen probes correctly identified the finding. Testing of polar bodies was performed via chromosome painting per methods described previously (11). Testing of blastomeres was performed via FISH on interphase chromosomes as previously described (11). Reciprocal translocation testing on blastomeres is completed by use of commercially available probes (Vysis) to identify translocated and nontranslocated portions of the chromosomes. All of the probe strategies allowed the discrimination of normal or balanced from unbalanced cells, but not of normal from balanced. Because translocation patients produce a large number of abnormal embryos (25), it would be uncommon to have only normal embryos for replacement. Most patients are comfortable with this approach. A minimum of three probes was used for reciprocal translocations. Typically, a combination of two probes placed distal to the breakpoints and one or two placed proximal to such are used. Robertsonian translocation testing on blastomeres is performed using enumerator probes for the chromosomes involved, and especially if the patient was 35 or older, other chromosomes such as X, Y, 8, 13, 14, 15, 16, 17, 18, 20, 21, or 22 were analyzed. The supplier s protocol with slight modifications (26) was followed for the first hybridization step. After the analysis of the first round of hybridization was completed and the slides were washed for 30 seconds, a second hybridization panel was applied. This step was repeated whenever a third round of hybridization was necessary. The tests used in this study did not detect uniparental disomy arising from balanced Robertsonian translocations. For this and other reasons, prenatal diagnosis was recommended after PGD. Robertsonian translocation carriers were informed that if prenatal diagnosis indicated a balanced translocation, UPD testing could be performed. Robertsonian translocation patients were informed that their risk of UPD was 1% or less if the balanced translocation was present. Informed consent for PGD was obtained from all patients, and outcome data was collected as a part of quality control procedures. Follow-up continued through pregnancy to postdelivery to obtain any prenatal and postnatal diagnoses. This review was determined to be exempt from institutional review board (IRB) approval. According to the Western IRB in Olympia, Washington, under common rule 45 CFR (b)(4), exemptions include research, involving the collection or study of existing data, documents, records, pathologic specimens, if these sources are publicly available or if the information is recorded by the investigator in such manner that subjects cannot be identified, directly or though identifiers linked to subjects. Preimplantation genetic diagnosis outcome was compared with published data in the literature. These datasets are summarized in Table Fischer et al. PGD aids RPL translocation carriers Vol. 94, No. 1, June 2010

3 TABLE 1 Outcomes of pregnancies achieved with and without PGD. Successful patient pregnancies (cumulative) Rate of miscarriage Timeframe to success With PGD % 15% 1.4 cycles (<3 mo) % 13% 1.4 cycles (<3 mo) % 5% 1.2 cycles (<3 mo) Without PGD % 64% unk a % 26% b 6y % 35% c 4.2 y Note: PGD ¼ preimplantation genetic diagnosis. 1: Lim et al. (2004), 2: This study, 3: Otani et al. (2006), 4: Sugiura-Ogasawara et al. (2004), 5: Goddjin et al (2004); 6: Stephenson and Sierra (2006). a Reciprocal translocation cases only; review of data over 16 years. b One termination of pregnancy (2%) secondary to Dandy-Walker malformation. c Reciprocal and Robertsonian translocation cases only; unbalanced form of translocation seen in some products of conception. RESULTS Chromosome Abnormalities The 192 patients with RPL underwent 272 cycles of PGD, of which only 16 used polar body biopsy and testing while the remainder (256) used biopsy and testing of blastomeres. Thirty-four percent of eggs tested revealed normal/balanced results. When divided by translocation type, 17% were normal for reciprocal translocations and 57% were normal for Robertsonian translocations (Table 2). Overall, 16% of embryos were normal/balanced, with 13% and 27% normal/balanced for reciprocal and Robertsonian translocations, respectively (Table 2). Testing of blastomeres allows for additional chromosomes to be studied for aneuploidy; hence, the lower percentage of normal embryos found at embryo biopsy. When tested, aneuploidy only is seen in 14% and 9% of embryos from Robertsonian and reciprocal translocation cases, respectively. Pregnancy Outcome Embryos were not transferred in 35% (94/272) of cycles most commonly because of there being no normal/balanced embryos available to transfer. Two of the 272 cycles had no transfer as all embryos were frozen. Thirty-nine percent (107/272) of cycles did not achieve pregnancy. Pregnancy was achieved in 25% (69/272) of the cycles started (Table 3) and in 39% of those with transfer (69/176). Of the 272 cycles, 220 were performed for reciprocal translocations and 52 for Robertsonian translocations (Table 3). Pregnancy rates per cycle started for these were 23% and 37%, respectively. Pregnancy rates per cycles with transfer were 38% and 41%, respectively (Table 3). Pregnancy rates are further defined for reciprocal and Robertsonian translocation cycles based on age (Tables 4 and 5) and previous pregnancy history (Tables 6 and 7). TABLE 2 Translocation carrier with three or more previous losses (192 patients). Robertsonian Reciprocal All Average age Eggs biopsied Normal/balanced % normal/balanced Embryos biopsied Normal/balanced % normal/balanced Fertility and Sterility â 285

4 A successful pregnancy was defined as a delivery or an ongoing pregnancy in the third trimester. Successful pregnancy rates per cycles started were 33% and 20% for Robertsonian and reciprocal translocations, respectively. Successful pregnancy rates per cycles with transfer were 38% and 33%, respectively (Table 3). Of the 192 patients, 68 (35%) patients obtained 69 pregnancies. Overall, 60/69 pregnancies were successful (87%) with 43/51 (84%) and 17/18 (94%) for reciprocal and Robertsonian translocation cases, respectively. Loss rates were therefore 13%, 16%, and 6%, respectively (Table 3). The 69 pregnancies were conceived from 99 cycles. This gives the average number of cycles to achieve pregnancy as 1.4. Comparison to non-pgd Data Sets Previous pregnancy history of the 192 patients at time of the first PGD cycle showed a total of 824 previous pregnancies. These resulted in 728 spontaneous abortions, 24 terminations, 2 stillbirths, and 69 liveborns. Pregnancy loss rate as determined by the number of spontaneous abortions and stillbirths was 88.5%. Only 8.3% (69/824) of the pregnancies resulted in live births. This compares unfavorably with the 13% loss rate and 87% success rate observed after PGD, although such comparison can be fraught with biases. Therefore, the RPL patients in this study were also compared with other datasets of patients with translocations not treated by PGD (Table 1). As Table 1 shows, the risk of miscarriage without PGD ranges from 26% to 64%, compared with only 13% in the present study. Successful pregnancy rates in the literature for non-pgd cycles range from 36% to 72% compared with 87% in the present study. Finally, although the rate of pregnancy success in the literature is 36% to 72% for non- PGD cases, these successes were achieved after 4 to 6 or more years of treatment. This is compared with our timeframe of only 1.4 IVF cycles (<3 months). DISCUSSION These data confirm the dramatic reduction in loss rates that PGD affords patients. Loss rates dropped to 13% overall, which is significant compared with the 88.5% loss rate before PGD. This is close to a sevenfold reduction in loss rate. Otani et al. (19) also found a dramatic reduction in loss. A 5.3% (1/19) loss rate after PGD for translocation in 33 patients who had lost all of their previous 117 pregnancies was seen. This reduction can obviously be assumed to be in part caused by the selection process in which patients with poor previous outcome are more likely to undergo PGD, even though they may have the same prognosis for the next pregnancy as patients with good previous reproductive history. For this purpose, we also compared these patients to other publications in the literature of patients with translocations untreated with PGD. These are summarized in Table 1. As it can be seen, the expected rate of miscarriage would be 26% to 64%, depending on the study, whereas we found an overall miscarriage rate of only 13%. More important, the success is achieved in only 1.4 IVF cycles compared with up to 6 years of treatment to achieve a 72% ongoing pregnancy rate without PGD (14). For translocation carriers with RPL that additionally have a female maternal age of 35 and over, waiting 6 years to achieve a viable pregnancy is not an option because fertility starts declining dramatically with advancing maternal age. High rates of abnormal embryos equate to fewer embryos for transfer and relatively low pregnancy rates (12, 25). The addition of aneuploidy testing to translocation cases does TABLE 3 Cycle outcomes. Robertsonian Reciprocal All Cycles Freeze No transfer Transfer Not pregnant Pregnant Pregnancy rate per cycle 37% 23% 25% Pregnancy rate per cycles with transfer 41% 38% 39% Number of pregnancies achieved Pregnancy success a 94% (17/18) 84% (43/51) 87% (60/69) Pregnancy lost 6% (1/18) 16% (8/51) 13% (9/69) Pregnancy success per total cycles 33% (17/52) 20% (43/220) 22% (60/272) Pregnancy success per cycles with transfer 38% (17/45) 33% (43/131) 34% (60/176) a Pregnancy reaching third trimester or child delivered per pregnancy achieved. 286 Fischer et al. PGD aids RPL translocation carriers Vol. 94, No. 1, June 2010

5 TABLE 4 Reciprocal translocation cycle outcomes per age group. <35 35 and over a Cycles Freeze 1 0 No transfer Transfer Not pregnant Pregnant Pregnancy rate 28% 16% per cycle Pregnancy rate per cycles with transfer 43% 30% a Only 11 (12%) of the 90 cycles of the 35 and over category were performed for women over 40 years of age. TABLE 6 Cycle outcomes: comparison of patients with and without children prior to PGD cycle. No Children Children Number of patients Cycles Freeze 2 0 No transfer Transfer Not pregnant Pregnant Pregnancy rate 26% 25% per cycle Pregnancy rate per cycles with transfer 38% 40% Note: PGD ¼ preimplantation genetic diagnosis. TABLE 5 Robertsonian translocation cycle outcomes per age group. <35 35 and over a Cycles Freeze 1 0 No transfer 3 3 Transfer Not pregnant 19 7 Pregnant 13 6 Pregnancy rate 36% 38% per cycle Pregnancy rate per cycles with transfer 41% 46% a Only 2 of the 16 (12.5%) cycles of the 35 and over category were performed for women over 40 years of age. further reduce the number of embryos available for transfer but should improve pregnancy outcomes if transfer occurs. Our population showed an average pregnancy rate of 25%, with 23% for reciprocal translocations and 37% for Robertsonian translocations. Pregnancy rates based on maternal age and previous pregnancy history are reported, but because of small sample sizes in each division, commentary on such is limited. Overall, higher rates tend to be seen in Robertsonian translocation PGD cases as, on average, there is typically at least one more embryo available for transfer. However, once pregnancy is achieved, the rate of success is high and similar between Robertsonian and reciprocal cases, indicating that it is only a matter of finding normal embryos to replace. Overall, the rate is 87%, with 84% and 94% for reciprocal and Robertsonian translocations. These success rates rival those seen in studies of pregnancies conceived naturally. Goddjin et al. (14) report that 25 of 41 known carriers of a chromosomal aberration had 72% (31/43) of pregnancies reach term with normal outcome or were ongoing. Stephenson and Sierra (23) report 71% of 58 pregnancies were liveborn for couples with chromosomal aberration. Sugiura-Ogasawara et al. (22) report an ongoing rate of 64% for Robertsonian translocation cases. However, they state only a 36% rate for reciprocal translocation cases. The latter of which is well below rates after PGD. Additionally, PGD can reduce time to success. Goddjin et al. (14) report an average timeframe to success of 6 years with a range of 2 to 9.8 years. Stephenson and Sierra (23) report an average of 4.2 years from time of onset of treatment and actual pregnancy (age 29.8 to 34 years). Our data demonstrates that the average number of cycles to achieve pregnancy was 1.4. Otani et al. (19) also found a short time to success with an average of 1.2 IVF cycles. These equate to a minimum timeframe to success of 3 months. Even if assuming more cycles are needed for success to compensate for lower pregnancy rates per cycle, couples undergoing IVF with PGD have the opportunity to undergo many cycles in one year if able. Length of time to success can therefore be dramatically shortened. The fact that PGD for translocations applied to carriers with RPL significantly reduced spontaneous abortion rates is in line with the reports of PGD for patients with idiopathic RPL, in which miscarriage is also significantly reduced (20, 21). Multiple investigators have reported high rates of aneuploidy in embryos of couples with RPL with rates approaching 80% (20, 27 29). The rate of abnormal embryos for translocation cases can surpass 80% as is seen in this study Fertility and Sterility â 287

6 TABLE 7 Cycle outcomes: comparison of patients with and without children prior to PGD cycle per translocation type. No children Children RECT ROBT RECT ROBT Cycles Freeze No transfer Transfer Not pregnant Pregnant Pregnancy rate per cycle 24% 31% 20% 60% a Pregnancy rate per cycles with transfer 39% 37% 36% 60% a Note: PGD ¼ preimplantation genetic diagnosis; RECT ¼ reciprocal translocation; ROBT ¼ Robertsonian translocation. a Sixty percent pregnancy rate based on only 10 cycles therefore may not be representative of population as a whole. and others (11, 12, 19, 25). Patients with RPL with and without translocations therefore find themselves in comparable risk groups for abnormal embryos. Both groups therefore benefit from PGD as such will allow for selection and transfer of chromosomally normal embryos. Stephenson and Sierra (23) report in their study that less than half of their patients with translocations had concomitant issues including antiphospholipid syndrome, luteal phase deficiency, factor V Leiden heterozygosity, and Crohn s disease. Their reported improvement in live birth rate comes when these patients are treated for said conditions and monitored closely through pregnancy after natural conception. However, 36% of the pregnancy losses in this group were found to have the unbalanced form of the chromosomal aberration seen in the parent. Patients undergoing IVF cycles with PGD receive thorough evaluations by their respective reproductive endocrinologists. Concomitant issues therefore are diagnosed and treated through the cycle and pregnancy as needed in addition to having the PGD for the translocation. Additionally, PGD all but eliminates the chance that losses, if they occur, are because of an unbalanced form of the familial translocation. This combination of treatment of concomitant issues and PGD seems the most thorough, and may be the best route to helping these patients have a successful pregnancy. When PGD is performed in our lab for translocation cases, patients must undergo a consult with a genetic counselor on staff. Patients report that PGD is being undertaken, as they can no longer physically and emotionally experience more pregnancy losses, nor do they want to waste more time in trying to create or complete their families. Patients believe PGD is the tool to help them find the embryos that are better able to give them success by reducing risk for losses or having an affected pregnancy all in a shorter time frame. When documenting patient commentary, one must recognize that patients seeking PGD may be a biased population of those emotionally or physically unable to continue conceiving naturally and comfortable with using ART. Likewise, a population of patients as seen in other studies may not desire, or have the option, to undergo PGD (14, 22, 23). Thus, we may be comparing two different populations regarding their desired treatment, although the difference in outcome may remain between treatments. PGD should be an option given to any patient with a translocation, therefore allowing the patient to decide the best course of action for herself. Advising patients to proceed with natural conception without considering PGD does not avoid risk for the patient. Patients conceiving and losing pregnancies are at risk for emotional and physical issues. Likewise, undergoing an IVF cycle is not without risk. Patients need to know all their options, and proceed with what they believe is their best choice. In summary, PGD for translocations undoubtedly reduces the pregnancy loss rate and can lead to success in a shorter amount of time than natural conception. Preimplantation genetic diagnosis, coupled with treatment of concomitant issues and surveillance by the reproductive endocrinologist, may be the best scenario for success for these couples. REFERENCES 1. Martin RH. Sperm chromosome analysis of two men heterozygous for reciprocal translocation: t(1;9)(q22;q31) and t(16;19)(q11.1;q13.3). Cytogenet Cell Genet 1992;60: Estop AM, Van Kirk V, Cieply K. Segregation analysis of four translocations, t(2;18), t(3;15), t(5;7) and t(10;12), by sperm chromosome studies and review of the literature. Cytogenet Cell Genet 1996;70: Escudero T, Lee M, Sandalinas S, Munne S. Female gamete segregation in two carriers of translocations involving 2q and 14q. Prenat Diagn 2000;20: Durban M, Benet J, Boada M, Fernandez E, Calafell JM, Lailla JM, et al. PGD in female carriers of balanced robertsonian translocations and reciprocal translocations by first polar body analysis. Hum Reprod Update 2001;7: Fischer et al. PGD aids RPL translocation carriers Vol. 94, No. 1, June 2010

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