Intracytoplasmic sperm injection of oocytes prior to extrusion of a polar body

(66ng/dL range: 2-45ng/dL) and normal free testosterone, 17-hydroxyprogesterone and DHEA-S levels. Peripheral blood karyotype was 46XX. She was referred to the Gynecology Service at the NIH to undergo additional testing to rule-out the presence of a contralateral ovotestis and gonadoblastoma in order to preserve her remaining ovary. Repeat endocrine studies now revealed a normal serum total testosterone level, and genetic testing for SRY was negative. A transvaginal ultrasound revealed a polycystic right ovary. Complete absence of an ovotestis with a gonadoblastoma is impossible to predict without a tissue diagnosis, a decision was made to perform Mohs micrographic surgery of the contralateral ovary in an attempt to preserve it. RESULTS: During the Mohs micrographic surgery, thin slices of ovarian tissue and a wedge resection were obtained from the hilum and sent for intra-operative frozen sections. Pathology revealed stromal cells with no evidence of testicular tissue or a gonadoblastoma, and no further dissections were performed. CONCLUSIONS: True hermaphroditism is characterized by the presence of both ovarian and testicular tissue. There may be an ovary on one side and a testis on the other, but more commonly one gonad is an ovotestis. Although the incidence of bilateral gonadoblastomas and gonadal neoplasia in 46 XX true hermaphrodites is rare, in the absence of a tissue diagnosis they cannot be ruled-out. Mohs micrographic surgery of the ovary represents a novel surgical approach to preserve fertility in these patients while making a tissue diagnosis. Supported by: In part, by the intramural research program of the Reproductive Biology and Medicine Branch, NICHD, NIH.

A-234 POSTPONEMENT AND PRESERVATION OF FERTILITY BY OOCYTE FREEZING: PRELIMINARY EXPERIENCE. J. G. Bromer, W. Somers, P. Patrizio. Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT. OBJECTIVE: While the techniques of oocyte cryopreservation are becoming increasingly refined, there is little data available on the subset of patients who have chosen to pursue this technology. Here, we analyze the epidemiology and demographic characteristics of our first 27 cycles of oocyte cryopreservation. DESIGN: Analysis of oocyte cryopreservation cycles at an academic center. MATERIALS AND METHODS: All patients were consented for an IRB approved protocol of oocyte cryopreservation using a slow freezing protocol. Patient and cycle charts were reviewed retrospectively for population characteristics, patient history, and cycle indications. RESULTS: A total of 27 cycles from 22 patients (mean age 34.5, range 14-40) were reviewed (see Table). These patients represented 45% of the number of initiated consultations. 41% (7/17) of the women had partners at the time of oocyte freezing. 44% (12/27) of cycles were performed for postponement of fertility, and 30% (8/27) for fertility preservation prior to initiation of gonadotoxic chemotherapy. 11% (3/27) of cycles were prompted by unexpected sperm unavailability. The remaining cycles involved egg freezing from anonymous donors for a pilot study to create a bank of cryopreserved oocytes.

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Patient Age

FUTURES IN FERTILITY: DEVELOPMENT OFA REPRODUCTIVE HEALTH PSYCHOEDUCATION PROGRAM FOR ADOLESCENT CANCER PATIENTS AND THEIR FAMILIES. J. Harned Adams, J. L. Sheeder, A. Cejka, J. Rhoda, E. A. Albano. Psychology, University of Colorado Denver, Denver, CO; The Children’s Hospital of Denver, Denver, CO; Center for Cancer and Blood Disorders, The Children’s Hospital of Denver, Denver, CO.

1

34

2 3 4 5 6 7 8 9 10 11

39 27 39 18 32 14 41 39 40 40

12 13 14 15 16 17 18 19 20 21 22

33 36 40 39 38 38 AD AD AD AD AD

OBJECTIVE: The Futures in Fertility Program (FFP) is designed to study the medical history, health habits, and fertility markers of adolescent patients and their parents following cancer diagnosis and the outcomes of their treatment on future reproductive health. FFP also aims to provide reproductive health and fertility preservation education. DESIGN: The focus of the current pilot study was to assess baseline levels of reproductive health knowledge and demand for services related to reproductive health. 28 patients (17 females; 11 males) and their parents have participated to date. MATERIALS AND METHODS: Patients and parents completed a demographics and health history, reproductive health knowledge survey, and a needs assessment survey, as well as measures associated with psychosocial outcomes. RESULTS: Reproductive Health Knowledge:Patients and parents have limited knowledge of reproductive health issues, with an average score of 67% of items correct. Reproductive Health Needs Assessment: Satisfaction with information received. Most patients were satisfied with information received regarding future fertility (82.1%) and menstruation (88.2%). Many patients reported dissatisfaction with information received about risk of future pregnancy and cancer recurrence (41.2%); impact of cancer on future pregnancy (29.4%) and risk of birth defects and cancer in future children (35.7% and 42.9%). Need for additional information. Patients report a need for additional information in the following domains: impact of treatment on fertility (44.4%) and menstruation (25%); risk of recurrence secondary to future pregnancy (56.3%); risk of complications in a future pregnancy secondary to current cancer treatment (81.3%); risk of birth defects in future children (77.8%) and risk of cancer to future children (74.1%). Preferences for support for reproductive health conncerns. 87.5% of patients reported a preference for receiving information about reproductive health via their oncology care team. 70.8% expressed an interest in fertility preservation. 50.0% reported an interest in support groups focused on reproductive health. CONCLUSIONS: Results indicate that AYA cancer patients and parents have limited reproductive health knowledge. Patients appear to be dissatisfied with information received surrounding this issue and are interested in receiving additional information and services related to reproductive health. These results are being used to guide the development of fertility services available to AYA cancer patients. Supported by: None.

FERTILITY & STERILITYÒ

Diagnosis Moral objection to embryo freezing Fertility Postponement Endometrial Cancer Fertility Postponement Sarcoma Sperm Unavailability Systemic Lupus Erythematosus Breast cancer Breast cancer Breast cancer Moral objection to embryo freezing Sperm Unavailability Fertility Postponement Fertility Postponement Sperm Unavailability Fertility Postponement Fertility Postponement Oocyte banking Oocyte banking Oocyte banking Oocyte banking Oocyte banking

No. No. Oocytes Partner Cycles Frozen Yes

1

9

No No No No Yes No No No No Yes

4 1 2 1 1 1 2 1 1 1

32 6 27 32 29 17 9 11 3 12

Yes No No Yes No No N/A N/A N/A N/A N/A

1 1 1 1 1 1 1 1 1 1 1

23 4 3 6 7 3 17 3 8 7 14

AD: Anonymous Donor. CONCLUSIONS: Oocyte cryopreservation is now being utilized for a wide range of indications, including fertility postponement and fertility preservation in the setting of gonadotoxic therapy. Unmarried patients with partners also select this procedure in addition to or instead of embryo cryopreservation. Moral objections to embryo cryopreservation also appear to play a role in the use of oocyte cryopreservation. Supported by: None.

FERTILIZATION A-235 INTRACYTOPLASMIC SPERM INJECTION OF OOCYTES PRIOR TO EXTRUSION OF A POLAR BODY. D. H. McCulloh, J. Goorbarry, A. Seungdamrong, P. G. McGovern, K. Ahmad. University Reproductive Associates, Hasbrouck Heights, NJ; UMDNJ - New Jersey Medical School, Newark, NJ; UMDNJ - New Jersey Medical School, Newark, NJ. OBJECTIVE: Retrieval of oocytes for in vitro fertilization (IVF) yields oocytes at different stages of maturity. While we hope to collect

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oocytes arrested at the 2nd meiotic prophase (MII), some oocytes are found with an intact germinal vesicle (GV) or lacking both the GV and a polar body (MI). We wished to determine the incidence of fertilization for these MI oocytes using intracytoplasmic sperm injection (ICSI). DESIGN: Retrospective Analysis of Laboratory Records. MATERIALS AND METHODS: Between 1/1/02 and 5/31/06, we performed ICSI for all oocytes from which the GV had disappeared (MI and MII) in the afternoon of oocyte retrieval day. The incidences of maturation and fertilization (presence of two pronuclei [PN]) were determined 16-20 hours after ICSI. RESULTS: During the period of the study, 299 patients underwent oocyte retrieval and had at least one MI oocyte. From these patients, 3888 oocytes were retrieved and prepared for ICSI. Of these oocytes, 2690 (69.2%) were MII oocytes and 459 (11.8%) were MI oocytes. An additional 546 (14.0%) had a germinal vesicle and were not injected. Each MI oocyte underwent ICSI with a motile sperm. For MI oocytes scored the next morning, 17 (3.1%) of the oocytes lysed following ICSI and 68 (12.5%) failed to extrude a polar body. The remaining 374 oocytes (81.5%) matured to the MII stage (extruded a polar body). Of the 374 mature oocytes, 147 (32.0%) had 2 PN (fertilized), whereas, 247 (53.8%) remained unactivated with no PN, and 65 (14.2%) had inappropriate numbers of PN [21 (4.6%)had only one PN and 44 (9.6%) had 3 or more PN]. In comparison, 2690 MII oocytes undergoing ICSI yielded 2046 (76.4%) with 2 PN, 259 (9.6%) inactivated with no PN, 146 (5.4%) lysed and 219 (8.1%) with inappropriate numbers of PN [97 (3.6%) had only one PN and 122 (4.5%) had 3 or more PN]. CONCLUSIONS: MI oocytes undergoing ICSI yielded a lower incidence of normally fertilized oocytes (32.0%) than did MII oocytes (76.4%). MI oocytes, while slightly less likely than MII oocytes to be lysed (3.1% vs. 5.4%, respectively), were also less likely to be activated by an injected sperm (R 1 PN) (46.2% vs. 89.8%, respectively). Presence of an injected sperm and extrusion of a polar body are insufficient to achieve activation or fertilization. ICSI of the MI oocytes yielded 147 additional embryos (7.2% increase) and an average of 0.49 (147/299) embryos per patient. It is not clear whether the increased number of available embryos per patient (from 6.97 to 7.37) impacted the incidence of pregnancy. Supported by: None.

A-236 CAN LATE FERTILIZED CONCEPTUSES IMPROVE CLINICAL OUTCOME? S. Hong, Q. V. Neri, T. Takeuchi, Z. Rosenwaks, G. D. Palermo. The Center for Reproductive Med & Infertility, Cornell University, New York, NY. OBJECTIVE: There is an optimal timing for fertilization and embryo cleavage to occur that often translates in a successful clinical outcome. However, it remains standard procedure in most laboratories to evaluate oocytes that fail to fertilize at the expected time. This study aims at determining whether it is still useful to evaluate these oocytes. DESIGN: A retrospective analysis of ICSI cycles where at least one oocyte displayed a delayed fertilization was carried out. Clinical outcome was compared with ICSI cycles displaying timely fertilization. MATERIALS AND METHODS: We analyzed 1,878 ICSI cycles between Jan 2007 to Mar 2008. For each cycle, we followed each oocyte from injection to transfer, identifying cases in which at least one oocyte showed delayed fertilization. Delayed fertilization is defined as the absence of pronuclei at 14-16 hrs post-injection, followed by the appearance of two pronuclei at 22-23 hrs. We compared fertilization and clinical pregnancy rates among cycles with delayed fertilization and those showing timely fertilization, controlling for differences in hCG administration, egg retrieval time and ICSI timing. Clinical pregnancy was defined as the presence of at least one FHB. RESULTS: The study group (n¼1,878) was comprised of an average maternal age of 36.1  6 years, an overall fertilization rate of 72.8% (10,913/14,990) and a clinical pregnancy rate of 30.9% (580/1,878). Out of 1,878 cycles, 107 (5.7%) had at least one oocyte showing delayed fertilization. Patients with mixed fertilization timing are listed in Table.

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Abstracts

TABLE 1. Outcome of cycles with different fertilization timing

Fertilization Timely Cycles MII oocytes Fertilization rate (2PN) Late fertilization eggs Adjusted fertilization Clinical pregnancy

1,702 13,570 9,972 (73.5) 0 9,972 (73.5)a 538 (31.6)b

Timely & delayed 101 1,037 745 (71.8) 138 883 (85.1)a 37 (36.6)b

P< 0.0001, bP< 0.01.

a

The group including mixed fertilization displayed a significantly a higher fertilization rate and clinical pregnancy rate. In 30 of those patients, 37 late fertilized embryos were transferred. In order to prove that those zygotes can generate a pregnancy, we identified six patients that received exclusively those zygotes and observed one ongoing pregnancy. CONCLUSIONS: Although the occurrence of late fertilization is remarkably low, routine assessment for the delayed appearance of pronuclei proved to be a meaningful policy. The observed benefit on clinical outcome implies the need for sustaining the practice of identifying these zygotes. Supported by: Institutional.

A-237 THE EFFECT OF SPERM SEPARATION ON SPERM CHROMATIN DECONDENSATION AND MOTILITY AT 0 AND 24 HOURS OF CULTURE. C. L. Bormann, A. M. Rocha, P. A. Hassun, E. L. A. Motta, P. Serafini, G. D. Smith. OB/GYN, University of Wisconsin, Madison, WI; Huntington Center for Reproductive Medicine, Sao Paulo, SP, Brazil; University of Michigan, Ann Arbor, MI. OBJECTIVE: The objective of this study was to determine which sperm separation technique yields the lowest level of sperm DNA fragmentation. DESIGN: Experimental. MATERIALS AND METHODS: Semen samples were collected, liquefied and divided into the following treatments: 1) Semen mixed v/ v with 2% H2O2 (control), 2) Fresh sample, 3) Sperm washed and centrifuged in HTFH (wash) 4) Swim-up (SU) from washed pellet of sperm 5) 45/90% ISolateÒ density gradient separation (DG) and 6) Density gradient separation followed by swim-up (DG/SU). Following sperm separation, DNA integrity was measured and 100,000 sperm/ml from treatments 2-6 were cultured in HTF þ 10% SSS at 37 C in 5% CO2 for 24 h. Following culture, sperm were analyzed using the sperm chromatin decondensation assay (SCD) and motility was recorded. Results of sperm DNA fragmentation and motility were analyzed using ANOVA and Turkey’s test for means. Significance was determined at P