OBJECTIVE: In 2001 we published our initial experience with microTESE and ICSI for the treatment of PPCA. The objective of the present study was to report our updated outcomes in this population to provide prognostically helpful information to physicians and affected couples. DESIGN: We queried our database of men who consecutively underwent microTESE at our institution from 1995-2007 and identified all patients who had previously received cytotoxic chemotherapy for retrospective analysis. MATERIALS AND METHODS: Oncologic data, pretreatment hormone profiles, testicular histology, and outcomes of microTESE-ICSI were reviewed. MicroTESE was performed by a single surgeon. Most patients without preoperatively available testicular histology underwent random testicular biopsy at the time of microTESE. Histology was classified by the most advanced spermatogenic pattern present. ICSI was performed using fresh spermatozoa. Embryos were transferred into the uterine cavity on the third day after microinjection. Clinical pregnancy was established by transvaginal ultrasonographic detection of a fetal heartbeat. RESULTS: 53 patients underwent microTESE-ICSI. Lymphoma was the most common malignancy (n=25, 47%), followed by leukemia (n=9, 17%), sarcoma (n=7, 13%), and testicular cancer (n=6, 11%). Mean patient and female partner ages were 34.8 years (range 22-54) and 32.5 years (range 21-43). The mean male serum follicle-stimulating hormone level was 22.4 mIU/mL (range 3.3-62.7). Testicular histology was available in 46 patients, 40 of whom had Sertoli cell only (SCO) pattern (87%) and 6 of whom had hypospermatogenesis (HS) (13%). MicroTESE was successful in 28 of 64 attempts (43% retrieval rate, mean 1.2 attempts per patient). Retrieval rates were significantly higher in men with HS (100%) than in men with SCO (33%) (p=.003, Fisher exact test). Clinical pregnancies following successful sperm retrieval were achieved in 15 of 28 couples (54%), which resulted in 6 live deliveries (21%). Four boys and 4 girls were delivered. CONCLUSIONS: The present study is the largest series reported to date of microTESE-ICSI in men with PPCA. Sperm are retrievable in greater than 40% of patients, despite the presence of SCO pattern on testicular biopsy in the overwhelming majority of cases. Patients with HS have a more favorable prognosis for sperm retrieval, but are not sufficiently common to justify routine diagnostic testicular biopsy prior to microTESE. Attempts at micro-TESE ICSI are worthwhile in this patient population.

Purpose: The aim of this study was to report the successful fertility treatment of men with Klinefelter syndrome using testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI). Methods: A total of 42 men with Klinefelter syndrome who underwent 54 TESE procedures were identified. Before TESE, patients with serum testosterone levels less than 15.6 nmol/liter were treated with an aromatase inhibitor. Sperm retrieval rates and results of ICSI, including fertilization and clinical pregnancy, were collected. Results: Mean pretreatment FSH and testosterone levels were 33.2 IU/liter and 9.8 nmol/liter. During medical therapy, the mean testosterone level rose to 17.0 nmol/liter (P < 0.01). Spermatozoa were found during 39 microdissection TESE procedures, on the day before, or day of oocyte retrieval during a programmed in vitro fertilization cycle. The sperm retrieval rate was 72% (39 of 54) per TESE attempt, and 29 of the 42 different men (69%) had adequate sperm found for ICSI. Thirty-three in vitro fertilization cycles yielded embryos for transfer in the 39 (85%) cycles with sperm retrieved. Eighteen clinical pregnancies have resulted in 21 live births [18 of 39 (46%)]. All children had a normal karyotype. Conclusion: TESE/ICSI is a successful intervention for the majority of patients with azoospermia and Klinefelter syndrome. Sperm retrieval and ICSI success in men with Klinefelter syndrome are comparable with other men with nonobstructive azoospermia treated at our center.

OBJECTIVE:To examine the results of a 3-year trial using blastocyst cryopreservation to limit multiple pregnancy and optimize overall pregnancy per cycle. DESIGN:Retrospective clinical evaluation of pregnancy rates after freezing and thawing human blastocysts. SETTING:Tertiary-care academic center. PATIENT(S):Seven hundred fifty-three different patients treated in 783 IVF cycles with blastocysts frozen from July 2000 to June 2003. INTERVENTION(S):Two thousand, two hundred fifty-nine blastocysts were frozen in cycles in which only blastocysts were cryopreserved (cycles with pronuclear stage oocytes or pre-embryos also cryopreserved were excluded from the analysis). Of these, 628 (27.6%) were thawed in 218 cycles. MAIN OUTCOME MEASURE(S):Pregnancy rate per cycle with thaw. RESULT(S):Four hundred seventy-nine (76.3%) blastocysts survived thawing, and 440 (92.0%) were transferred after exhibiting evidence of survival (most commonly, blastocoele reexpansion). In cycles with a thaw, 211 (96.8%) of 218 underwent intrauterine transfer. An average of 2.09 blastocysts was transferred per replacement. One hundred twenty-five (59.2%) clinical pregnancies were established, which included 23 sets of twins and 5 triplet gestations. Two sets of monozygotic twins were identified after the replacement of a single thawed blastocyst (1.6%). The age of the patient at the time of cryopreservation (<37 years) was an important factor in the establishment of clinical and ongoing pregnancy. The mode of ovarian stimulation, replacement method, and whether blastocysts were frozen on day 5 or day 6 of development did not demonstrate clinical significance. CONCLUSION(S):Cryopreserved and thawed blastocysts demonstrated a similar potential for implantation when compared with fresh pre-embryos on day 3. On the basis of these results, the blastocyst stage of development appears to be optimal for clinical freeze-thaw trials.

One or more viable preembryos (PE) often remain following transfer on Day 3 (D3) post-harvest. In our laboratory, these extra PE are cultured in sequential media for the purpose of cryopreserving any good quality blastocysts (BL) formed on Day 5 or Day 6 (D5/6). This has resulted in an augmentation of fresh clinical pregnancy rates for many patients. It is not known if the pregnancy status following D3 transfer is indicative of a patient's chances of becoming pregnant in a subsequent thaw cycle using sibling BL. The objective of the current study was to determine if the pregnancy and implantation potential of thawed human BL derived from patients who had attained pregnancy in their D3 fresh cycle differed from those patients with a negative D3 pregnancy result. A retrospective analysis of 173 BL thaw transfers from 2002–2004 was performed. Patients included in the analysis had one or more sibling BL cryopreserved following extended culture after D3 transfer. Thaw cycles were categorized into two groups based on the pregnancy status of their D3 fresh cycle: Group 1 included patients (N=73) who achieved pregnancy in their D3 cycle, while Group 2 included patients (N=100) who had a negative pregnancy result on D3. Endpoints included post-thaw blastocyst survival rate, clinical and ongoing pregnancy rates, and implantation rate. Stimulation protocols, sperm preparation techniques, PE and extended culture methods, and BL freezing and thawing methods used in the Cornell IVF laboratory have been described in detail elsewhere. Following the transfer of fresh PE on D3 post-harvest, any remaining PE were placed in extended culture using C2 medium and evaluated daily. Blastocysts of good quality were subsequently cryopreserved on D5 or D6. The transfer of thawed BL was performed in either a natural or programmed cycle. Day 5 cryopreserved BL were thawed and cultured overnight in C2 medium prior to transfer, while D6 cryopreserved BL were thawed on the day of transfer. Data were analyzed by chi-square or t-test where appropriate. The overall clinical pregnancy rate of BL thaw patients in this analysis was high (87/173; 50%). There were no significant differences between Group 1 and Group 2 patients when comparing post-thaw blastocyst survival rates (74% and 79%). Similarly, clinical pregnancy rates (52% and 49%), ongoing/delivered pregnancy rates (44% and 37%) and implantation rates (39% and 35%) were not different between the two groups. No significant group differences were found in mean patient age and the number of blastocysts replaced at transfer. Blastocysts resulting from the extended culture of sibling PE following D3 transfer can be cryopreserved on D5/6 and subsequently thawed, resulting in high rates of implantation and pregnancy. The pregnancy status of the D3 fresh cycle has no effect on the potential success of subsequent thaw cycles using sibling blastocysts.

The implantation process involves complex interactions and synchrony between the embryo and the endometrium. Culturing embryos on an autologous endometrial coculture system has been demonstrated to be beneficial for patients who have had multiple failed IVF cycles or a history of sub-optimal embryo morphology. However, at present, there is no clear understanding of the mechanisms resulting in the benefits of increased pregnancy and implantation rates (1). Recently, an L-selectin-mediated adhesion system was proposed as an initial step of the implantation process, wherein L-selectin located on the trophoblast will interact with a uterine carbohydrate selectin ligand during the endometrial receptive period (2). We used immunofluorescent techniques to detect L-selectin and its ligand in an in vitro endometrial coculture system. Human endometrial cultures and deselected day 7 embryos from consenting IVF patients were analyzed using monoclonal antibodies to L-selectin and its corresponding ligand. Human endometrial cell cultures were obtained from luteal endometrial biopsies and assessed for the presence of the selectin ligand and the specific cell type in which it is expressed. Anti-selectin ligand antibodies (HECA-452) were used to determine the presence of the ligand in extended in vitro cultures of individual or mixed stromal and glandular endometrial cells. Cell-type specific antibodies, vimentin for stromal and cytokeratin for glandular cells, were also used to identify the types of cells expressing the ligand. In addition, the anti-L-selectin antibody (DREG-56) was used on the limited number of deselected human day 7 embryos to detect its presence. All specimens were analyzed using immunofluorescence techniques. The L-selectin ligand is expressed in human endometrial cells, even when cultured in vitro. Due to their high plasticity, it is often difficult to identify the different endometrial cell types when grown in culture. However, we have circumvented these challenges by using cell-type specific antibodies that recognize the glands from stromal cells. The selectin ligand is expressed, albeit not uniformly, on the surface of cytokeratin-positive glandular endometrial cells, while vimentin-positive stromal cells lacked any signal. Finally, L-selectin is homogeneously detected on the surface of human trophectoderm. The L-selectin system can be detected and studied in human endometrial cultures in vitro. The expression of the L-selectin ligand is restricted to the glandular endometrial cells in a non-uniform pattern. Additionally, L-selectin is expressed on the surface of human embryo trophectoderm. These results suggest that L-selectin may act as a mediator in embryo-endometrial recognition and, intriguingly, could be used in the future as a marker for endometrial receptivity. (1) Fertil Steril (2002) 77:1209–1213; (2) Science (2003) 299:405r–408

While an IVF cycle is considered successful when a pregnancy is achieved, it is optimal to reduce the risk of multiple gestations. To prevent this risk, the ASRM guidelines suggest reducing the number of preembryos (PE) transferred back to the patient, particularly on day 3. The decision regarding which preembryos should be selected for embryo transfer (ET) can be difficult, as the preembryos chosen for transfer on day 3 are generally selected based upon their morphological appearance. The choice of blastocyst for day 5 or day 6 ET is much easier since they have undergone their own self-selection during the additional days of culture. We assessed the value of our day 3 preembryo selection criteria in terms of its ability to predict whether a chosen preembryo would eventually develop to the blastocyst stage and be selected for ET or cryopreservation on days 5 or 6. Fifty-one day 5 or day 6 IVF transfer cycles from October 2003 to February 2004. On day 3, preembryos that would have been transferred if the patient were undergoing day 3 ET were grouped separately (referred to as pre-selected preembryos, PSPE) and cultured alongside the remainder of the preembryos until days 5 or 6. Patients were selected for blastocyst transfer (days 5/6 ET) based on the number of fertilized oocytes, developmental stage at 24/26 hrs, and overall morphology and cell number on day 3. From day 3 to days 5/6, preembryos were cultured in separate groups based on their overall quality. The PSPEs were chosen based on their developmental speed and morphology and were cultured separately from the rest of the preembryo cohort. On days 5 or 6, the overall best quality and most advanced blastocysts were selected for ET. Of the remaining ones, only expanded blastocysts with good morphology were cryopreserved. We analyzed the predictive value of the day 3 pre-selection criteria for blastocyst development, ET, and cryopreservation on day 5/6. Transfer of ninety-nine day 5/6 blastocysts from 51 study patients (1.9 PE/ET) resulted in pregnancy and implantation rates of 68% and 55%. One hundred and fifty-nine preembryos were pre-selected as if they were undergoing day 3 ET (PSPE) (3.1 PE/ET). Of these, 54 (34%) were chosen for ET, 51 (32%) were cryopreserved, and 54 (34%) were arrested and discarded by day 6. In total, 105/159 (66%) PSPEs were either transferred or cryopreserved. In 31% of the cases where two blastocysts were transferred (48 transfers), both had developed from PSPEs. One of the two blastocysts was a PSPE in 51% of the cases, and, 18% of the PSPEs were not chosen at all. If we sum ET and cryopreservation numbers, we selected all of the blastocysts that had developed from a PSPE 45% of the time. Only 4% of the times were no PSPE selected for ET; in the remaining cases at least one was pre-selected. Preembryo selection based on developmental speed and morphology, particularly on day 3, still has limitations. However, using our selection criteria, we can correctly pre-select 66% of the preembryos for day 5/6 ET or cryopreservation. These results are encouraging as we observe comparable pregnancy (70%) and implantation (over 50%) results, as well as a reduction in multiple gestations in matched day 3 and day 5 IVF patients with only two preembryos transferred.

ObjectiveRecent studies suggest that the phenotypic manifestation of paternal genomic abnormalities likely does not occur before implantation, but it is unclear if the paternal genome affects the development of a day-3 embryo to a blastocyst (BL). The goal of this study is to determine if male factor infertility influences the progression of an embryo to BL.DesignCase-control study.Materials and methodsPatients who underwent day-5 embryo transfer from June 1999 to December 2003 at an academic IVF center were separated into two groups: male factor infertility (MF) and non-male factor (NMF). MF was defined as having a history of a MF plus a total motile sperm count (TMC) of < 20 X 106. To minimize age as a confounder, patients with age ≥ 40 years were excluded from NMF. Embryos on day-3 were separated into the following groups: (A) > 8 cells, (B) 6–8 cells, (C) 4–5 cells, and (D) < 4 cells. Progression of every embryo in each group to day 5 was noted and compared between groups. Patient variables and IVF outcomes were analyzed using Chi-square or T-test.ResultsThere was no difference between MF and NMF groups with regards to mean age, BMI, previous failed and successful IVF cycles, amount of stimulation medication used, # oocytes harvested, # mature oocytes, # 2PN, # embryos transferred (ET), # pregnancy sacs, % patients with BL transfer on day-5, % embryos progressing to BL overall, # frozen BL, fertilization rate, and implantation rate. Between MF and NMF, statistically significant differences were noted with regards to the % ICSI cycles, TMC, and the # of 1PN, 3PN, and degenerated embryos. Furthermore, a significantly lower percentage of embryos within group A (> 8 cells) progressed to BL in the MF group as compared to the NMF group (32.6% vs. 44.7%, respectively, p ≤ 0.05). Progression to BL was not different in groups B, C, and D between MF and NMF. These results are presented in table 1. In addition, there was a trend for a decreased % to BL when comparing group A to group B in the MF group (32.6% vs. 42.4%, respectively, p ≤ 0.1) vs. no difference in the NMF group (44.7% vs. 43.9%, respectively, p ≫ 0.05).Conclusions ObjectiveRecent studies suggest that the phenotypic manifestation of paternal genomic abnormalities likely does not occur before implantation, but it is unclear if the paternal genome affects the development of a day-3 embryo to a blastocyst (BL). The goal of this study is to determine if male factor infertility influences the progression of an embryo to BL. Recent studies suggest that the phenotypic manifestation of paternal genomic abnormalities likely does not occur before implantation, but it is unclear if the paternal genome affects the development of a day-3 embryo to a blastocyst (BL). The goal of this study is to determine if male factor infertility influences the progression of an embryo to BL. DesignCase-control study. Case-control study. Materials and methodsPatients who underwent day-5 embryo transfer from June 1999 to December 2003 at an academic IVF center were separated into two groups: male factor infertility (MF) and non-male factor (NMF). MF was defined as having a history of a MF plus a total motile sperm count (TMC) of < 20 X 106. To minimize age as a confounder, patients with age ≥ 40 years were excluded from NMF. Embryos on day-3 were separated into the following groups: (A) > 8 cells, (B) 6–8 cells, (C) 4–5 cells, and (D) < 4 cells. Progression of every embryo in each group to day 5 was noted and compared between groups. Patient variables and IVF outcomes were analyzed using Chi-square or T-test. Patients who underwent day-5 embryo transfer from June 1999 to December 2003 at an academic IVF center were separated into two groups: male factor infertility (MF) and non-male factor (NMF). MF was defined as having a history of a MF plus a total motile sperm count (TMC) of < 20 X 106. To minimize age as a confounder, patients with age ≥ 40 years were excluded from NMF. Embryos on day-3 were separated into the following groups: (A) > 8 cells, (B) 6–8 cells, (C) 4–5 cells, and (D) < 4 cells. Progression of every embryo in each group to day 5 was noted and compared between groups. Patient variables and IVF outcomes were analyzed using Chi-square or T-test. ResultsThere was no difference between MF and NMF groups with regards to mean age, BMI, previous failed and successful IVF cycles, amount of stimulation medication used, # oocytes harvested, # mature oocytes, # 2PN, # embryos transferred (ET), # pregnancy sacs, % patients with BL transfer on day-5, % embryos progressing to BL overall, # frozen BL, fertilization rate, and implantation rate. Between MF and NMF, statistically significant differences were noted with regards to the % ICSI cycles, TMC, and the # of 1PN, 3PN, and degenerated embryos. Furthermore, a significantly lower percentage of embryos within group A (> 8 cells) progressed to BL in the MF group as compared to the NMF group (32.6% vs. 44.7%, respectively, p ≤ 0.05). Progression to BL was not different in groups B, C, and D between MF and NMF. These results are presented in table 1. In addition, there was a trend for a decreased % to BL when comparing group A to group B in the MF group (32.6% vs. 42.4%, respectively, p ≤ 0.1) vs. no difference in the NMF group (44.7% vs. 43.9%, respectively, p ≫ 0.05). There was no difference between MF and NMF groups with regards to mean age, BMI, previous failed and successful IVF cycles, amount of stimulation medication used, # oocytes harvested, # mature oocytes, # 2PN, # embryos transferred (ET), # pregnancy sacs, % patients with BL transfer on day-5, % embryos progressing to BL overall, # frozen BL, fertilization rate, and implantation rate. Between MF and NMF, statistically significant differences were noted with regards to the % ICSI cycles, TMC, and the # of 1PN, 3PN, and degenerated embryos. Furthermore, a significantly lower percentage of embryos within group A (> 8 cells) progressed to BL in the MF group as compared to the NMF group (32.6% vs. 44.7%, respectively, p ≤ 0.05). Progression to BL was not different in groups B, C, and D between MF and NMF. These results are presented in table 1. In addition, there was a trend for a decreased % to BL when comparing group A to group B in the MF group (32.6% vs. 42.4%, respectively, p ≤ 0.1) vs. no difference in the NMF group (44.7% vs. 43.9%, respectively, p ≫ 0.05). Conclusions

Reducing the incidence of multiple births after IVF is a major challenge. The most efficient means to accomplishing this goal remains reducing the number of embryos transferred (ET). However, selecting the patient population in whom to limit the number of ET without compromising clinical pregnancy rates (PR) is a challenge. Thus, as part of this effort, we determined if having available embryos for cryopreservation in fresh cycles is predictive of higher likelihood of multiple pregnancies. Retrospective cohort study. We reviewed records from a 5 year period of IVF patients undergoing day 3 ET who had embryos available for day 3 cryopreservation (Group A, 434 cycles) and compared them to patients undergoing day 3 ET without cryopreservation (Group B, 3192 cycles). Cycles with freezing on other days were excluded. We compared clinical and multiple PR and stratified patients by age and number of ET. Statistical analysis was performed using Fisher's exact test, and p<.05 was considered statistically significant. Patients in group A were more likely than patients in group B to establish clinical pregnancies: for patients age <34, 75.9% vs. 56.7%, (p<.001); age 34–39, 64.3% vs. 50.5%, (p<.001); and age 40–41, 57.9% vs. 34.9%, (p<.01). Patients in group A were also more likely than patients in group B to have multiple pregnancies: age <34, 46.9% vs 37.9%, (p<.001); age 34–39, 34.3% vs. 28.6% (p<.001); and age 40–41, 23.7% vs. 7.8% (p<.001). Tables IA-IC demonstrate the clinical pregnancy and multiple PR for each age group stratified by group A vs. B and by number of ET. *p=.30, Group A, 2 vs. 3 ET.†p=.22, Group A, 3 vs 4 ET.‡ p=.62, Group A, 4 vs 5 ET. The availability of embryos for cryopreservation on day three is predictive of a higher likelihood of clinical and multiple PR. Specifically, our data suggests that transferring 2 embryos in patients age <34 and 3 embryos in patients age 34–39 who have embryos available for day three cryopreservation affords an excellent clinical PR while diminishing the risk of multiple pregnancy.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Advances in reproductive medicine have dramatically changed the scope of male infertility patients who can be treated with assisted reproduction. Sperm obtained from the testis can be effectively used to effect fertilization and subsequent pregnancies. However, the simple view that “all sperm are equal” for intracytoplasmic sperm injection (ICSI) and that sperm quality will not affect the success rates or offspring is sorely naive. Recent, adequately powered studies have shown that at least two different populations of patients may have testicular sperm used for ICSI. 1 Park Y.S. Lee S.H. Song S.J. et al. Influence of motility on the outcome of in vitro fertilization/intracytoplasmic sperm injection with fresh vs. frozen testicular sperm from men with obstructive azoospermia. Fertil Steril. 2003; 80: 526-530 Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar These include men with normal sperm production (and quality) with obstruction (obstructive azoospermia) and patients with severely impaired sperm production, in whom the sperm quality is so severely impaired that sperm do not survive to reach the ejaculate (nonobstructive azoospermia). Clear differences exist in the ability to use the spermatozoa from the two different sources for assisted fertilization. The pregnancy rates are expectably greater, with lower spontaneous abortion (miscarriage) rates when testicular sperm from men with obstructive (versus nonobstructive) azoospermia are used. 2 Palermo G.D. Schlegel P.N. Hariprashad J.J. et al. Fertilization and pregnancy outcome with intracytoplasmic sperm injection for azoospermic men. Hum Reprod. 1999; 14: 741-748 Crossref PubMed Scopus (255) Google Scholar For men with nonobstructive azoospermia, the spermatozoa are so severely impaired in quality that they will not survive transit out of the body, so there is no reasonable expectation that they should reliably survive the multiple physical and biochemical insults that occur during cryopreservation and subsequent thawing.

Blastocyst transfer (BL-ET), as opposed to day 3 preembryo transfers, represents a beneficial treatment for IVF patients because it allows for optimal embryo selection, which in turn results in high pregnancy (PR) and implantation (IM) rates with a concomitant reduction in the incidence of multiple gestations (only one to two blastocysts transferred). One of the main questions is if there is a significant difference in developmental potential of day 5 (D5) blastocysts as compared to day 6 (D6). Further, if such a difference exists, is it also reflected when comparing D5 and D6 frozen/thawed cycles? To answer these questions and determine which day, if either, is most advantageous for preembryo transfer (ET), we compared D5 and D6 PR and IM outcomes in fresh and frozen BL-ET cycles. A retrospective analysis of clinical PR and IM rates in 548 fresh BL-ET (D5 and D6) and 359 frozen BL-TR (frozen either D5 or D6) was performed. Patients were divided into groups based on transfers with fully developed blastocysts and transfers with only morulae (no blastocysts transferred). Patients cultured to D5 or D6 solely due to medical indications or slow development were kept as a separate group. IVF patients with an adequate number of good quality preembryos on day 3 were selected for BL-ET on D5 (n=532) or D6 (n=16). Blastocysts with good morphology were frozen on D5 (n=115 thawed cycles) or D6 (n=225 thawed cycles). The patient groups were subdivided as follows: D5 fresh, with blastocysts transferred (n=496) or morulae transferred (n=36); D6 fresh (15/16 with blastocysts transferred); D5 frozen; D6 frozen; medical/slow. Statistical analysis was performed using Fisher or Chi Square tests. Clinical PR and IM rates were significantly higher in fresh BL-ET as compared to frozen/thawed: fresh BL-ET cycles (including both D5 and D6) demonstrated 68% clinical PR vs. 49% frozen/thawed, and 52% IM vs. 23% frozen/thawed rates (P=0.0001). Taken independently, the D5 and D6 fresh cycles showed no significant differences for clinical PR (69% vs. 56%) or IM (52% vs. 43%). Significantly higher PR and IM rates were observed in patients with transferred blastocysts as opposed to transferred morulae in fresh D5 cycles (71% vs. 33% and 56% vs. 17% respectively, p=0.0001). In addition, patients with only morulae on D5 achieved greater clinical PR results (56% vs. 33%, NS) and significantly better IM rates (43% vs. 17%, p=0.0006) if they were left in culture and transferred on D6 (D6 blastocyst vs. D5 morula/ET). There were no differences between D5 and D6 frozen/thaw cycles for PR (50% vs. 48%) or IM (23% vs. 23%). Patients undergoing D5/D6 ETs due to medical indications or slow preembryo development (not selected for BL-ET by virtue of good growth rate and morphology) had poor outcomes, with only an 8% PR rate and 6% IM rate. Blastocyst transfer, whether fresh or frozen, represents a good treatment option for IVF patients, although fresh BL-ET did demonstrate better pregnancy and implantation results as compared to frozen/thawed. Overall, we observed no differences between D5 and D6 fresh ET. In addition, cryopreservation of blastocysts on D5 or D6 showed no differences in pregnancy and implantation rates in subsequent thawed cycles. Finally, we recommend that preembryos with only morulae on day 5 be left in culture an additional day; this allows for improved blastocyst selection, which then results in a higher rate of pregnancy and implantation.

Objective: Men with nonobstructive azoospermia (NOA) may have sperm retrieved from testicular tissue for use in intracytoplasmic sperm injection (ICSI). While cryopreserved sperm from men with obstructive azoospermia have been used for ICSI as effectively as fresh sperm in this population, it is unclear if the low numbers of sperm retrieved from men with NOA survive the freeze/thaw process sufficiently for use in ICSI. We have assessed the effect of freeze/thaw on sperm from men with NOA and present the results of ICSI using frozen-thawed testicular sperm in NOA. Design: Retrospective chart review. Materials and Methods: Patients with NOA who had frozen testicular tissue thawed on the day of an ICSI cycle were identified and patient demographics recorded. The post-thaw processed sample was evaluated for motility using pentoxifylline stimulation. For samples with adequate numbers of viable sperm, ICSI was applied using these spermatozoa. If inadequate, repeat testicular sperm extraction (TESE) was performed. Outcomes for ICSI were recorded, relative to the source of spermatozoa used. Donor sperm were used if repeat TESE was unsuccessful, and the couple desired use of this backup sample. Results: A total of 96 cycles were evaluated in which frozen testicular specimens were thawed for use in an ICSI cycle. Motile spermatozoa were documented for all specimens prior to cryopreservation. Of 31 cycles performed with frozen/thawed sperm alone, a fertilization rate of 57% and clinical pregnancy rate of 39% were observed. In the remaining 65 cycles, the thawed sample did not contain sufficient motile sperm for injection. Repeat TESE was performed in 61 cycles. For the 50/61 (82%) with enough motile sperm found on repeat TESE to inject all oocytes, the fertilization rate with use of fresh sperm was 59% and clinical pregnancy rate 46%. Mean maternal age ± SD was 35.5 ± 5.0 years for the frozen group and 33.7 ± 5.3 for the group in which fresh testicular sperm were used for ICSI. Of the remaining 15 patients for whom the thawed specimen was inadequate for ICSI, 2 couples used donor sperm for ICSI and 2 couples used a combination of thawed and donor sperm, while 11 had inadequate sperm to inject all oocytes after repeat TESE. Inadequate spermatozoa were found in 11/61 (18%) cases of repeat TESE, requiring use of donor sperm (n = 3), nonmotile frozen/thawed sperm (n = 4), a combination of donor and frozen/thawed sperm (n = 2), a combination of donor and fresh TESE sperm (n = 1), or cancellation of the ICSI cycle (n = 1). In total, 65 of 96 ICSI cycles (68%) could not be performed with a frozen/thawed testicular specimen and required an immediate alternative sperm source. Conclusion: Although the majority of men with NOA may have successful TESE, most testicular specimens from men with NOA that are frozen (68%) are not useable for ICSI, and an alternative sperm source, such as repeat TESE or donor sperm, must be immediately available. Performance of TESE intended for cryopreservation only for a future ICSI cycle is limited in utility, as only 32% of specimens may have sufficient motile sperm available following thaw. For men with NOA, TESE is ideally planned concurrently with oocyte retrieval.

Objective: The role that individual multinucleated blastomeres (MNBs) play in further preembryo (PE) development is unclear, most reports complicated by an inconsistent presence and unclear origin of this particular finding. Here, we attempted to determine if the presence of MNBs, as a single variable, is associated with reduced developmental capacity. Design: Retrospective study. Materials and Methods: A total of 2016 preembryos from 200 patients were included in the analysis. One would expect to observe 2 cells on Day 1 (26 hrs after insemination or 24 hrs after ICSI), 4 cells on Day 2, and 8 cells on Day 3. Blastomere number and presence/absence of MNBs were evaluated daily. Preembryos were also analyzed for MNB size and distribution. Results: On Day 1, the frequency of MNBs in cleaved PE was low (9.5%). The highest frequency of MNBs was observed in 2-cell preembryos on Day 2 (34%), while a lower incidence was seen in greater cell stages on the same day (22%, 6%, and 12% for 3-, 4-, and 5-cells respectively). Interestingly, developmental rates did not appear to be compromised when MNBs were noted on Days 1 or 2; no differences in average cell number on Day 3 were observed between MNB and non-MNB groups. Growth rate on Day 2, however, did impact Day 3 findings. A difference in average cell number on Day 3 was associated with the cell stage on Day 2, significantly better quality preembryos developing from Day 2 PE with 4 and 5 cells as compared to 2 cells (7.5 vs. 5.4 cells on Day 3 for advanced and slower cleavage on Day 2, respectively). In addition, the average cell number on Day 3 in PE developing from 2-cell, with or without MNB on D1, was comparable to 4 and 5 cells on Day 2. PE persisting at the 2-and 3-cell stage on D2 (highest frequency of MNBs) developed primarily from slower developing conceptuses on D1 (90% from pronuclear stage). Four-cell PE on D2 developed equally from 2-cell (31%), sygamy (41%) and pronuclear (27%) stages on D1, regardless of MNB status. The size and distribution of MNB noted at various cell stages did not influence developmental outcome. Conclusion: The results suggest that the appearance of MNBs, used as a single variable, does not appear to influence subsequent development and confirms that MNBs are not consistently featured during sequential cell divisions. On the other hand, regular rate of development has the greatest positive influence on subsequent PE development in vitro.

Objective: Preimplantation genetic diagnosis (PGD) procedures are commonly used to check for specific genetic diseases and chromosomal abnormalities. This study was undertaken to determine if preembryo morphology on day 3 and rate of blastocyst development on day 5 are associated with PGD results. Design: Retrospective analysis of PGD cycles tested for aneupolidy from January 2000 to March 2003. Materials and Methods: Six-hundred nineteen preembryos from seventy-six PGD cycles were divided into two main groups: Normal (preembryo diagnosed as being free of studied chromosomal abnormality), and Abnormal (diagnosed as carrying a chromosomal disorder). Each group was subdivided into three subgroups according to preembryo morphology and development on days 3 and day 5. The mean age of all patients in this study was 38.88 ± 5.05. Data were compared using Chi-Square statistical analysis. PGD was performed on day 3. Fluorescent In Situ Hybridization (FISH) techniques, using probes for chromosomes X, Y, 13, 18, and 21 (and often, 15, 16, and 22), were used for all diagnoses. Occasionally, probes for chromosome 2, 9, 10, 11, 17, or 20 were added for individual cases. Results: One hundred ninety-six preembryos (32%) were classified as normal, four-hundred and three (65%) were shown to be abnormal, and twenty (3%) had unconfirmed diagnoses. Results are summarized in the table below. When a single chromosome abnormality was analyzed in the abnormal group, results demonstrated that preembryos with XXY, XYY, monosomies for 16, 21, 22, and trisomies for 13, 15, 16, 18, and 22 all were capable of developing into blastocysts by day 5. Tabled 1 Conclusion: Normal preembryos, as diagnosed using common FISH probes, appear to develop at faster rates than abnormal ones on days 3 and 5. However, the finding that more than 50% of chromosomally abnormal preembryos are viable and reached morula or blastocyst stages suggests the importance of aneuploidy screening.

Objectives: High order multiple pregnancies (HOMP) are the single largest cause of poor obstetrical outcome and neonatal difficulties. Some patients choose to undergo selective reduction of one or more conceptuses to reduce the risks associated with HOMP. Other patients choose no clinical intervention and deliver all babies, while a third group of patients experience a spontaneous pregnancy loss. The objectives of this study were: 1) to determine the incidence of HOMP and spontaneous loss in our IVF program and 2) to identify factors affecting the choice of HOMP patients to undergo selective reduction. Design: A retrospective analysis of 650 cycles resulting in HOMP from 1995–2002 was performed. Patient cycles were grouped as follows: patients delivering all babies (DEL ALL); patients choosing to undergo selective reduction (SLRE); and patients experiencing a spontaneous loss but who still delivered at least one baby (SPONT). Factors studied included patient age, number of previous IVF cycles, previous pregnancy and previous pregnancy loss. Materials and Methods: Preembryos (PE) were cultured in either human tubal fluid (prior to 2000) or a sequential C1/C2 media (after 2000). Transfer of PE was performed on Day 3 post-harvest. High order multiple pregnancies were documented based on the presence of 3 or 4 fetal heart beats at the first ultrasound, approximately 5 weeks after transfer. Data were analyzed by student's t-test or chi-square where appropriate. Results: The overall rate of HOMP in our program was 6.0% (650/10,784 transfers). Of these, 25 patients (including 6 SLRE patients) miscarried (3.8%) resulting in a loss of all conceptuses. To date, 68 patients have ongoing pregnancies while 557 delivered at least one baby. Of the 557 deliveries, 191 (34.3%) delivered all babies, 218 (39.1%) underwent SLRE, and 148 (26.6%) experienced a spontaneous loss of one or more conceptuses with delivery. When these data were stratified by age, clear group differences were observed. In older HOMP patients (40 years of age and higher), over half (53%) experienced a spontaneous loss of at least one baby, double the rate found in younger patients (P<.001). The highest percentage of patients choosing to deliver all babies (43.8%; P<.01) was seen in the youngest patient group (<34 years of age), while the percentage of SLRE patients was highest in the 34–39 age group (P<.01). One other factor appeared to play an important role in determining if patients chose to undergo SLRE. A significantly lower percentage of patients chose SLRE when at least one pregnancy loss had occurred in previous treatment cycles (24.6%) as compared to the DEL ALL (34.2%) group (P<.05). In addition, the percentage of SPONT patients was high (35.0%) when a previous loss had occurred. No group differences were seen when comparing patients based on previous successful births or previous number of IVF attempts. Conclusion: Patients with HOMP face difficult clinical decisions that may affect subsequent obstetrical outcome. In our program, approximately equal proportions of HOMP patients chose to undergo SLRE or deliver all babies. The age of the patient and the occurrence of a previous pregnancy loss appeared to be important factors in determining if a patient chose to undergo SLRE or to deliver all babies.

Objective: To compare clinical pregnancy and implantation rates between frozen/thawed preembryos and blastocysts (frozen on Day 3 vs. frozen on Days 5 or 6).Design: Retrospective analysis of transfers carried out in a university, hospital-based setting.Materials/Methods: Preembryos frozen and thawed between April 1995 and March 2002, and blastocysts frozen and thawed between July 2000 and March 2002 were included in the analysis. In an effort to compare homogeneous populations, cycles with Day 1 pronuclear oocytes (bias toward upfront freezing of all fertilized oocytes) or those with combination freezing groups (subsequent mixed transfer) were excluded from analysis. Day 3 preembryos were cryopreserved after exposure to increasing concentrations of 1,2 propanediol using a slow-freeze protocol. Blastocysts were similarly cryopreserved, but glycerol was used as the cryoprotectant. Thaws were carried out using a 30°C waterbath. Most blastocysts were selected for freezing after being deemed unsuitable for either replacement or freezing on Day 3. It is the policy of the Cornell program to culture these deselected preembryos until days five or 6, documenting growth and morphology daily.Results: Findings are summarized in the table. Patient age did not differ between the preembryo and blastocyst groups (34.8 ± 4.8 and 34.8 ± 4.6 years, respectively). However, the number of conceptuses transferred after thaw was significantly higher in the Day 3 group (3.7 vs. 2.1; p <0.0001). Despite having fewer conceptuses replaced, clinical pregnancy and implantation rates were significantly greater from frozen/thawed blastocysts.TableSurvival, Clinical Pregnancy, and ImplantationDay FrozenCycles With FreezeCycles With ThawSurvived/Thawed (%)Clin Preg/Tr (%)Implanted/Tr (%)383440878.8a: NS;37.4b: p < 0.005;14.9c: p < 0.00015 or 63825875.6a: NS;60.3b: p < 0.005;39.7c: p < 0.0001Chi Square Analysisa : NS;b : p < 0.005;c : p < 0.0001 Open table in a new tab Conclusions: Blastocyst freezing trials were initiated by the Cornell program in July, 2000 as an alternative to Days 1 and 3 protocols employed historically. Although fewer blastocysts were frozen per cycle with freezing (4.0 blastocysts vs. 6.0 preembryos), the overall proportion of patients having cryopreservation did not change after blastocyst freezing was adopted (19% vs. 20%). Despite that most frozen blastocysts developed from deselected preembryos, a significant increase in pregnancy and implantation was observed from these conceptuses after thaw. No differences in clinical pregnancy or implantation were noted between blastocysts frozen on Day 5 or Day 6. We conclude that once an acceptably proficient level of blastocyst culture has been achieved, freezing at this stage optimizes a patient's chances for becoming pregnant after thaw.Supported by: The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University. Objective: To compare clinical pregnancy and implantation rates between frozen/thawed preembryos and blastocysts (frozen on Day 3 vs. frozen on Days 5 or 6). Design: Retrospective analysis of transfers carried out in a university, hospital-based setting. Materials/Methods: Preembryos frozen and thawed between April 1995 and March 2002, and blastocysts frozen and thawed between July 2000 and March 2002 were included in the analysis. In an effort to compare homogeneous populations, cycles with Day 1 pronuclear oocytes (bias toward upfront freezing of all fertilized oocytes) or those with combination freezing groups (subsequent mixed transfer) were excluded from analysis. Day 3 preembryos were cryopreserved after exposure to increasing concentrations of 1,2 propanediol using a slow-freeze protocol. Blastocysts were similarly cryopreserved, but glycerol was used as the cryoprotectant. Thaws were carried out using a 30°C waterbath. Most blastocysts were selected for freezing after being deemed unsuitable for either replacement or freezing on Day 3. It is the policy of the Cornell program to culture these deselected preembryos until days five or 6, documenting growth and morphology daily. Results: Findings are summarized in the table. Patient age did not differ between the preembryo and blastocyst groups (34.8 ± 4.8 and 34.8 ± 4.6 years, respectively). However, the number of conceptuses transferred after thaw was significantly higher in the Day 3 group (3.7 vs. 2.1; p <0.0001). Despite having fewer conceptuses replaced, clinical pregnancy and implantation rates were significantly greater from frozen/thawed blastocysts. Chi Square Analysis Conclusions: Blastocyst freezing trials were initiated by the Cornell program in July, 2000 as an alternative to Days 1 and 3 protocols employed historically. Although fewer blastocysts were frozen per cycle with freezing (4.0 blastocysts vs. 6.0 preembryos), the overall proportion of patients having cryopreservation did not change after blastocyst freezing was adopted (19% vs. 20%). Despite that most frozen blastocysts developed from deselected preembryos, a significant increase in pregnancy and implantation was observed from these conceptuses after thaw. No differences in clinical pregnancy or implantation were noted between blastocysts frozen on Day 5 or Day 6. We conclude that once an acceptably proficient level of blastocyst culture has been achieved, freezing at this stage optimizes a patient's chances for becoming pregnant after thaw. Supported by: The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University.

Objective: The objective of the study was to evaluate a simple preembryo scoring system for predicting a patient's chances for becoming pregnant. Design: A retrospective analysis of day 3 preembryo transfers collected over a one-year period in a University, hospital-based system was performed. Donor oocyte recipients and patients over 40 years of age were excluded from the data set. Materials/Methods: Before transfer, preembryos were assigned a score based on blastomere number and morphology. For each preembryo transferred, the number of blastomeres was multiplied by a fragmentation score (1 = >20% fragmentation; 2 = 11–20%; 3 = ≤10%). A cumulative preembryo transfer score was calculated by adding the individual score(s) of each preembryo transferred. For data analysis, the mean score of each transfer group (1 transferred, 2 transferred, 3 transferred, or 4 transferred) was calculated. A breakpoint of -1 standard deviation (SD) was found to provide the greatest differences in subsequent pregnancy and implantation results. Interestingly, scores above the mean or greater than +1 SD of the mean were not associated with increasingly higher pregnancy or implantation rates. It was determined that the optimal breakpoint values (mean -1 SD) for cumulative preembryo scores when 1, 2, 3, or 4 preembryos were transferred were 16, 34, 54, and 73, respectively. Data were analyzed using the Fisher's exact test with significance at p <0.05. Results: The number of patients, patient age, clinical pregnancy rate (CP), and implantation rate (IR) for preembryo scores below and above the breakpoint of -1 SD of the mean is detailed in the table below. These data are categorized based by the number of preembryos transferred. The clinical pregnancy and implantation rates of patients having a cumulative preembryo score greater than -1 SD of the mean was significantly higher than in patients with a score less than -1 SD of the mean when 2, 3, or 4 preembryos were transferred. This trend was also observed in single preembryo transfers, although differences were not significant because of low numbers. aNo. TransfNo. CyclesLess than −1 SDNo. CyclesGreater than −1 SDAvg AgeCP/ Transf (%)IR/ Transf (%)Avg AgeCP/ Transf (%)IR/ Transf (%)11235.116.7a= NS;16.7e= NS;6034.230.0a= NS;30.0e= NS;22933.534.5b= p < 0.05;20.7f= p < 0.05;15333.159.5b= p < 0.05;37.6f= p < 0.05;36432.340.6c= p < 0.0001; chi-square analysis.19.8g= p < 0.0001; chi-square analysis.40732.167.3c= p < 0.0001; chi-square analysis.37.6g= p < 0.0001; chi-square analysis.47435.848.7d= p < 0.05;17.6h= p < 0.0005;44736.262.0d= p < 0.05;27.5h= p < 0.0005;a = NS;b = p < 0.05;c = p < 0.0001; chi-square analysis.d = p < 0.05;e = NS;f = p < 0.05;g = p < 0.0001; chi-square analysis.h = p < 0.0005; Open table in a new tab Conclusions: A simple cumulative preembryo transfer score based on blastomere number and percent fragmentation proves effective in predicting acceptable or reduced chances for clinical pregnancy when -1 SD of the mean is used as a breakpoint value. Pregnancy and implantation were not enhanced with scores above the mean or above +1 SD of the mean, suggesting that day 3 preembryos with good morphology and greater than average blastomere number do not contribute at higher rates to positive outcomes. Supported by: The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University.

Objective: The assisted hatching (AHA) procedure is based on the hypothesis that a making an artificial hole in the zona pellucida promotes hatching and implantation potential, particularly in older women. This study was undertaken to determine if assisted hatching truly improves clinical pregnancy and implantation rates for in-vitro fertilization (IVF) patients 40 years old and older. Design: Retrospective analysis of patients in a university, hospital-based IVF clinic from January 2000 to April 2002. Materials/Methods: Six-hundred twenty-eight (628) IVF cycles (505 different patients) were divided into two main groups: cycles with AHA (+) and cycles without AHA (-). Each group was then subdivided into three subgroups related to the patient's previous history [Subgroup 1 = first-time patients producing good quality-preembryos (<15% fragmentation and no excessively-thick zonae) where some were offered (or requested) AHA because of age and some were not; subgroup 2 = patients with a previous history demonstrating pregnancy success and where AHA was carried out or not carried out to match the previous successful cycle; and, subgroup 3 = patients with a previous history demonstrating pregnancy failure where the opposite use of AHA was applied in order to change treatment options. Assisted hatching was performed using acidified Tyrode's solution. A 20–25 m opening was created in the zonae pellucidae of consenting patients undergoing transfer on day 3. Blastocyst transfer patients (day 5 transfers) were excluded from the analysis. Data were compared using Chi-Square statistical analysis. Results: Between all groups and subgroups, there were no differences in mean age or mean number of preembryos transferred. Results are summarized in the table below. ∗ aGroupNo. CyclesAgeAvg. No. Preembryos TransferredClinical Preg/ TransferImplanted/ Preembryo TransferredAHA+28341.9 ± 1.44.1 ± 1.631.8%10.9%1+13241.7 ± 1.43.8 ± 1.633.3%12.6%2+11542.2 ± 1.44.4 ± 1.730.4%9.4% versus3+3641.5 ± 1.54.4 ± 1.330.6%10.2%AHA−34541.5 ± 1.44.0 ± 1.634.8%11.7%1−24141.5 ± 1.43.9 ± 1.535.3%11.8%2−3841.2 ± 1.04.1 ± 1.644.7%15.6% P < 0.05 in favor of no hatching3−6641.6 ± 1.33.9 ± 1.727.3%8.9%∗ a versus∗ b P < 0.05 in favor of no hatching Open table in a new tab Conclusions: Based on these results, we conclude that pregnancy and implantation rates were not different between hatched versus non-hatched groups, or between any clinical pregnancy subgroup. Assisted hatching does not appear to improve pregnancy or implantation in women over the age of 40 years with good-quality preembryos. Supported by: The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University.

Objective: Using strict criteria, it has been demonstrated that a higher percentage of normal forms (% NF) correlates positively with pregnancy rates following classical insemination procedures (Kruger, et al., 1986). Nevertheless, the value of using strict criteria for predicting ICSI outcomes is still unclear. The purpose of this study was to determine if a correlation exists between percent normal forms and fertilization/pregnancy/implantation rates after ICSI. Design: Retrospective analysis of ICSI and insemination cycles carried out in a university-hospital based setting. Materials/Methods: Two thousand six-hundred and eighteen (2,618) ICSI cycles and 1091 insemination cycles were evaluated (January 1995 to December 2001). Patient variability was controlled by excluding treatment cycles where the female partner was greater than age 38, developed fewer than four mature oocytes, underwent preimplantation genetic diagnosis, or required coculture because of multiple previous poor outcomes. Parameters of the WHO Laboratory Manual, Fourth Edition were followed for analysis of sperm concentration and motility. The methods described by Kruger, et al., were followed for determination of % NF. ICSI and insemination cycles were divided into four groups as follows: 0%, 1 to 4%, 5 to 14%, and greater than 14%. The three parameters evaluated among the groups were fertilization rate per inseminated or injected oocyte (2PN), pregnancy rate/transfer (PR), and implantation rate as defined by gestational sacs/preembryos transferred (IR). Results: ICSI and insemination groups were remarkably similar for maternal age and average number of preembryos replaced (no significant differences between groups). In ICSI cycles, a significant trend was noted for increased fertilization as samples improved in morphology up to 14% NF. Contrary to this finding, insemination cycles yielded their greatest increase in fertilization above the 14% NF breakpoint. Generally, normal fertilization was achieved at higher rates in ICSI cycles as compared to insemination cycles, but this difference may be attributed to the fact that only mature oocytes are injected during ICSI procedures while all oocytes are exposed to sperm during insemination procedures, thus skewing the comparison. While pregnancy and implantation rates followed similar trends during both ICSI and insemination treatment, a significantly poorer IR was observed in insemination cycles using sperm with 0% NF. Pregnancy was also much reduced in this group but did not reach significance due to the lower number of cycles undergoing insemination procedures with 0% NF. Conclusions: Fertilization rates may increase slightly when working with semen samples exhibiting better morphology, both in ICSI and insemination cycles. PR and IR are relatively stable across morphology categories with ICSI, unlike the correlative trend seen at the lowest morphology category during insemination. This may be because the most normal-appearing sperm are handpicked for injection during ICSI procedures. Supported by: CRMI. Table TableICSI/Insemination vs. % NF0% NF1–4% NF5–14% NF14% NFICSI 2PN (%)71.5a77.2b79.2c76.6Insem 2PN (%)69.869.3d70.9e75.5fICSI PR (%)58.960.660.962.5Insem PR (%)43.3g63.8h56.760.4ICSI IR (%)30.8i32.132.434.0Insem IR (%)20.4j35.7k30.7l33.0mAcross: a v b; a v c = p < 0.0001 b v c; d v f; j v k = p < 0.005 e v f; g v h; j v l; j v m = p < 0.05 Down: b v d; c v e = p < 0.0001 i v j = p < 0.05 Open table in a new tab Across: a v b; a v c = p < 0.0001 b v c; d v f; j v k = p < 0.005 e v f; g v h; j v l; j v m = p < 0.05 Down: b v d; c v e = p < 0.0001 i v j = p < 0.05

Objective: Coculture of embryos with monolayers of AECC has been shown to improve outcome for patients with multiple implantation failures after In Vitro Fertilization (IVF). Presumably, this effect is mediated through the production of trophic factors expressed by these helper cell lines. TGF-2 and EGF are known to be produced by the endometrium (Giudice LC. Trends Endocrinol Metab, 1995; 6:60), and receptors have been found on embryos (Lim H. Dev Biol, 1998;204:97–110). The specific aim of this study was to determine if TGF-2 and EGF levels as measured in the supernatants of conditioned media (CM) of embryos cocultured in AECC is predictive of IVF outcome.Design: CM from the ECC cells exposed or non-exposed to human embryos was collected in 27 patients and assayed for the presence of TGF-2 and EGF and correlated with outcome.Materials/Methods: During a luteal phase biopsy (5–10 days after LH surge) made prior to the treatment cycle, glandular (G) and stromal (S) endometrial cells were isolated by enzymatic digestion and separated based on differential sedimentation rates. These cells were cryopreserved, then plated as a 50%/50% combination of G and S cells prior to embryo exposure. The conditioned medium (CM) was changed every 2 days. Embryos were randomly grown on ECC or conventional media if more than 6 oocytes normally fertilized. Otherwise, all embryos were grown on AECC. TGF-2 and EGF levels were measured utilizing an immunoenzymetric assay. Background levels of TGF-2 and EGF were also determined from media alone (Hams F-10 supplemented with 15% patient serum). Statistics included Wilcoxon signed rank test, Mann-Whitney U test and Chi-square.Results: The average age of the patients studied was 36.5 ± 3.5 years. Mean numbers of mature oocytes, fertilized embryos, and embryos transferred were 8.3 ± 4.1, 5.9 ± 3.5, and 3.5 ± 1.4 respectively. Clinical pregnancy rate was 37% and implantation rate was 14%. Exposure or non-exposure to an embryo did not result in differing levels of TGF-2 and EGF in the CM. Embryos grown on ECC demonstrated a significant improvement in number of blastomeres and fragmentation (frag) when compared to embryos grown in conventional media without ECC (6.7 ± 1.3 vs. 5.6 ± 1.2 blastomeres and 17.6% ± 9.3 vs. 26.4% ± 9.8 frag; p <0.05). TGF-2 and EGF levels in the CM were not associated with embryo quality or pregnancy outcome.Conclusions: We have demonstrated a significant improvement in blastomere number and fragmentation with ECC. TGF-2 and EGF are expressed by the cells in the AECC. The presence of TGF-2 and EGF in the CM was not associated with embryonic development or outcome.Supported by: None. Objective: Coculture of embryos with monolayers of AECC has been shown to improve outcome for patients with multiple implantation failures after In Vitro Fertilization (IVF). Presumably, this effect is mediated through the production of trophic factors expressed by these helper cell lines. TGF-2 and EGF are known to be produced by the endometrium (Giudice LC. Trends Endocrinol Metab, 1995; 6:60), and receptors have been found on embryos (Lim H. Dev Biol, 1998;204:97–110). The specific aim of this study was to determine if TGF-2 and EGF levels as measured in the supernatants of conditioned media (CM) of embryos cocultured in AECC is predictive of IVF outcome. Design: CM from the ECC cells exposed or non-exposed to human embryos was collected in 27 patients and assayed for the presence of TGF-2 and EGF and correlated with outcome. Materials/Methods: During a luteal phase biopsy (5–10 days after LH surge) made prior to the treatment cycle, glandular (G) and stromal (S) endometrial cells were isolated by enzymatic digestion and separated based on differential sedimentation rates. These cells were cryopreserved, then plated as a 50%/50% combination of G and S cells prior to embryo exposure. The conditioned medium (CM) was changed every 2 days. Embryos were randomly grown on ECC or conventional media if more than 6 oocytes normally fertilized. Otherwise, all embryos were grown on AECC. TGF-2 and EGF levels were measured utilizing an immunoenzymetric assay. Background levels of TGF-2 and EGF were also determined from media alone (Hams F-10 supplemented with 15% patient serum). Statistics included Wilcoxon signed rank test, Mann-Whitney U test and Chi-square. Results: The average age of the patients studied was 36.5 ± 3.5 years. Mean numbers of mature oocytes, fertilized embryos, and embryos transferred were 8.3 ± 4.1, 5.9 ± 3.5, and 3.5 ± 1.4 respectively. Clinical pregnancy rate was 37% and implantation rate was 14%. Exposure or non-exposure to an embryo did not result in differing levels of TGF-2 and EGF in the CM. Embryos grown on ECC demonstrated a significant improvement in number of blastomeres and fragmentation (frag) when compared to embryos grown in conventional media without ECC (6.7 ± 1.3 vs. 5.6 ± 1.2 blastomeres and 17.6% ± 9.3 vs. 26.4% ± 9.8 frag; p <0.05). TGF-2 and EGF levels in the CM were not associated with embryo quality or pregnancy outcome. Conclusions: We have demonstrated a significant improvement in blastomere number and fragmentation with ECC. TGF-2 and EGF are expressed by the cells in the AECC. The presence of TGF-2 and EGF in the CM was not associated with embryonic development or outcome. Supported by: None.

Objective: To determine if controlled ovarian hyperstimulation (COH) with FSH compared to FSH/LH in IVF impacts upon blastocyst transfer pregnancy rates. Design: A retrospective analysis of IVF cycles from January 2000 through December 2001 of patients undergoing blastocyst transfers at an academic center. Materials/Methods: Between January 2000 and December 2001, 248 patients underwent blastocyst transfer following COH with either pure FSH or FSH/LH. Patients were divided into a younger age group (<36 years old) and an older group (≥36 years old). Variables such as female's age, total ampules of gonadotropins utilized, estradiol level at the time of hCG administration, number of oocytes retrieved, number of mature eggs, fertilization rate, number of embryos transferred, implantation rate, and clinical/ongoing pregnancy rate per transfer were compared in the FSH vs. FSH/LH groups. Statistical analysis was performed using student's t test or chi squared test where applicable. Results: In the younger group, the use of FSH vs. FSH/LH did not reveal any statistically significant differences in any of the parameters. In the older group, the use of pure FSH was associated with a higher number of oocytes retrieved(p = .037) whereas FSH/LH usage yielded a higher fertilization rate (p = .038). However, implantation rates and clinical/ongoing pregnancy rates were not significantly different (see table). TableFSH vs FSH/LH Blastocyst ResultsVariablesFSH Group <36 years oldFSH/LH Group <36 years oldFSH Group ≥36 years oldFSH/LH Group ≥36 years old#Patients119473547Age (years)30.3 ± 3.532.1 ± 2.538.5 ± 2.038.1 ± 1.0E2@hCG (pg/ml)1953.5 ± 643.22013.8 ± 824.22225.2 ± 587.71988.0 ± 852.1#Oocytes14.6 ± 5.0315.6 ± 5.7217.9 ± 3.8315.5 ± 5.45#Mature oocytes13.7 ± 4.314 ± 5.515 ± 3.6ap = .03713 ± 4.4ap = .037Fertilization rate (%)78 ± 1476.8 ± 1378.1 ± 14bp = .03882 ± 12bp = .038#Blastocysts transferred2.03 ± 0.202.00 ± 0.202.23 ± 0.602.2 ± 0.54Total ampules24.7 ± 8.026.0 ± 7.128.0 ± 9.131.0 ± 10.0Implantation rate (%)56.658.544.943.7Pregnancy rate/transfer (%)75.872.368.667.7Values are means ± standard deviation.a p = .037b p = .038 Open table in a new tab Values are means ± standard deviation. Conclusions: This study demonstrates that the use of FSH alone vs. FSH/LH does not appear to confer any difference on blastocyst transfer pregnancy rates. Supported by: none.