Effectiveness of sperm washing by discontinuous density gradient centrifugation to remove antibodies bound to sperm membrane

P-646 Wednesday, October 22, 2014

P-648 Wednesday, October 22, 2014

TELOMERE LENGTH DETERMINATION IN HUMAN SPERMATOGENESIS. M. Brassesco,a R. Lafuente,a G. Lopez-Granollers,a C. Brassesco,a J. Benet,b J. Ribas-Maynou,c A. Garc
ıa-Peir
o.c aHuman Reproduction and Infertility Center (CIRH), Corachan Center, Barcelona, Spain; bBiologia Cel$lular, Fisiologia i Immunologia, Universitat Aut onoma de Barcelona, Bellaterra, Barcelona, Spain; cCentro de Infertilidad Masculina y An
alisis de Barcelona (CIMAB), Bellaterra, Barcelona, Spain.

INEQUITY EXISTS BETWEEN MALE AND FEMALE INFERTILITY COVERAGE IN STATE INSURANCE LAWS. J. M. Dupree, R. Dickey, G. M. Langille, R. Ramasamy, J. Kovac, L. I. Lipshultz. Scott Department of Urology, Baylor College of Medicine, Houston, TX.

OBJECTIVE: The objective of the present study was to assess the relative telomere length in human spermatogenesis and testicular spermatozoa. DESIGN: From 15 human testicular biopsy samples, telomere length was analyzed in all human spermatogenesis stages and testicular spermatozoa through Fluorescent In Situ Hybridization using Peptide Nucleic Acid probes (PNA-FISH). The fluorescence intensity was used as a relative measure of telomere length. MATERIALS AND METHODS: Human testicular biopsies had received a previous hypotonic treatment before Carnoy fixation. Afterwards, PNAFISH technique was performed on slides. This technique consists of a DNA denaturation step followed by a 1-hour hybridization with PNA probes at room temperature. A minimum of 30 cells per stage were captured and the fluorescence intensity of each telomere was analyzed using TFL-Telo software. RESULTS: Mean telomere intensity was expressed as arbitrary units
standard deviation: 1189
1118 in interphase; 1257
865 in leptotene; 1431
1170 in zygotene; 1006
834 in pachytene; 994
744 in metaphase I; 1022
624 in metaphase II, and 2109
2050 in testicular sperm. CONCLUSION: Results suggest a uniform telomere length during meiosis, interestingly, telomere length showed about 2-fold increase in testicular sperm cell. These results suggest an important telomere function during spermiogenesis. Supported by: Centro de Infertilidad y Reproducci
on Humana and Instituto de Salud Carlos III (FIS, Project: PI11/00630).

P-647 Wednesday, October 22, 2014 EFFECTS OF INTRA UTERINE GROWTH RESTRICTION ON TESTES. S. Demirbag,a U. Harkness,a J. Parvadia,a D. Alaee,a T. Crombleholme.a aPediatric Surgery, Gulhane Military Medical Academy, Ankara, Turkey; bPediatric Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. OBJECTIVE: There is a recognized association between the intrauterine growth restriction (IUGR) and chronic diseases such as, type 2 diabetes, hypertension and heart disease. We have previously shown that placental gene transfer of Insulin-like Growth Factor-1 (IGF-1) corrects growth in models of IUGR. We hypothesized that there is a relationship between IUGR and subfertility in males. To test this hypothesis we examined the effects of surgically induced IUGR on postnatal testes histology. DESIGN: An experimental model. MATERIALS AND METHODS: Time mated Sprague-Dawley rats were treated on day 18 of a 23-day gestation; Control (n¼4), Uterine Artery Ligation (UAL, n¼3), Adult male rats were sacrificed at 36 weeks and testicles were harvested. Morphometric analysis was performed was using Seminiferous Tubule Area (STA), Germinal Layer Area (GLA),as well as quantification of Sertoli cell, Spermatogonia, and Spermatocyte counts per tubule. Statistical analysis was performed using ANOVA. RESULTS: UAL resulted in a significant reduction in STA compare to control (0.035
.01 vs,0.44
.08mm2,p¼0.001). UAL resulted in a significant lower GLA compared to control rats (0.01
.001 vs. 0.3
.01mm2, p¼0.001). Sertoli cell counts were significantly lower in UAL compare to control (11.11
3 vs. 33.77
1.8, p¼0.007 Spermatocyte counts were significantly lower in UAL compare to control (103.77
9.10vs229.33
21.71, p¼0.01). Spermatogonia counts were significantly lower in UAL compare to Control (39.66
11.54 vs. 66.66
2.99, p¼0.03). CONCLUSION: UAL mediated IUGR results in sub-fertility in adult males. These results provide proof of concept that placental gene therapy may be an effective strategy for correction of adult disease related to IUGR such as male sub-fertility. These results may become implemented for other adult diseases associated with IUGR. Supported by: Supported by American Association of Obstetricians and Gynecologists Foundation.

FERTILITY & STERILITYÒ

OBJECTIVE: With the Affordable Care Act silent about federal mandates for insurance coverage for infertility work-up and treatment, the issue will be left to individual states. A minority of states currently have laws that address insurance coverage for infertility evaluation or treatment. While several studies have analyzed female infertility coverage in these states, none have evaluated coverage for male infertility. Malefactor infertility alone accounts for 20% of all infertility and is associated with an increased risk of cardiovascular disease, cancer, and early mortality. We hypothesize that male infertility evaluation and treatment is disproportionately excluded in state laws that address insurance coverage for infertility. DESIGN: We identified and compared states with laws or codes related to insurance coverage for infertility. MATERIALS AND METHODS: We used the National Conference of States Legislatures and the National Infertility Association to identify state laws and performed a primary review and analysis of the laws or codes to specifically identify coverage for male infertility services. RESULTS: There are 16 states with laws addressing insurance coverage for infertility (AR, CA, CT, HI, IL, LA, MD, MA, MN, MT, NJ, NY, OH, RI, TX, WV). Of the 16 states, 10 (62.5%) clearly mandate evaluation or treatment for female infertility. Only 6 states (37.5%) clearly mandate male factor evaluation or treatment. In two states (WV, MT), infertility coverage is listed only as part of covered basic health care services but is not further defined. In CA and TX, infertility coverage must be offered to employers, but employers may elect to include or exclude infertility coverage for their employees. Three states (MA, NY, NJ) specifically exempt coverage for elective sterilizations, including vasectomy reversal. CONCLUSION: Despite the Centers for Disease Control and Prevention’s and the American Society for Reproductive Medicine’s recommendations to include both male and female partners in infertility evaluation and treatment, only 37.5% of states with laws concerning infertility clearly include coverage for the male partner. Excluding men from infertility coverage risks missing opportunities to diagnosis serious health conditions in men, correct reversible causes of infertility, and provide cost-effective treatments that can downgrade the intensity of intervention required for the couple to achieve a pregnancy.

P-649 Wednesday, October 22, 2014 EFFECTIVENESS OF SPERM WASHING BY DISCONTINUOUS DENSITY GRADIENT CENTRIFUGATION TO REMOVE ANTIBODIES BOUND TO SPERM MEMBRANE. D. T. Schneider, C. M. Feij
o, S. Verza Junior, S. C. Esteves. Androfert, Campinas, S~ao Paulo, Brazil. OBJECTIVE: We evaluated the effectiveness of sperm washing using thediscontinuous two-layer density gradient centrifugation method to remove antisperm antibodies attached to the sperm surface. DESIGN: Prospective study. MATERIALS AND METHODS: We prospectively enrolled sixty-sixmenwith unexplained infertility seeking evaluation. Each patient delivered one semen specimen for the study.We determined antisperm antibodies (ASA) levels using the direct immunobeads test (IBT). Specimens were classified into two groups according to the pre-washing levels of antibody-bound spermatozoa: group 1 (low ASA levels, IBT<20%; n¼54) and group 2 (high ASA levels, IBTR20%; n¼12). Sperm washing was carried out using the discontinuous two-layer colloidal density gradient centrifugation method. Pre- and post-wash levels of antisperm antibodies were compared in the groups. RESULTS: The pre- and post-wash percentage of spermatozoa with antisperm antibodies attached to their surface was 11% and 6.5% in group 1 (mean difference ¼ 40%; p<0.01), and 30% and 19.5% in group 2 (mean difference¼36.8%, p¼0.02), respectively. The effectiveness of density gradient centrifugation in removing antisperm antibodies was not different between the groups, but individual variation from -52.3% to -3.9%was observed.

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Table 1. Effect of density gradient centrifugation on ASA levels before and after sperm washing in patients with low (IBT<20%) and high (IBT >20%) ASA.

% Antisperm antibodies Pre-wash Post-wash Percent change p value

Group 1 (IBT <20%)

Group 2 (IBT >20%)

11.0 (7.0; 15.0) 6.5 (4.0; 11.0) -40.0 (-57.1; -16.6) <0.01

30.0 (24.0; 32.5) 19.5 (13.0; 27.5) -36.8 (-52.3; -3.9) 0.02

p value <0.001 <0.001 0.75

Data reported as median and 95% confidence interval. Percent change calculated as median of individual changes. Wilcoxon rank sum test used to compare pre- and post-wash ASA levels, as well as ASA levels between the groups. CONCLUSION: Sperm washing by density gradient centrifugation is an overall effective method to remove antibodies bound to sperm membranes, regardless of the levels of ASA in the neat semen. Due to an inter-individual variation in the effectiveness of the method, we recommend that each patient be tested before applying sperm washing by density gradient centrifugation in intrauterine insemination. P-650 Wednesday, October 22, 2014 EFFECT OF PATERNAL AGE ON BLASTOCYSTS ANEUPLOIDY RATES. D. Das, T. H. Taylor, J. Patrick, J. Crain, N. Teaff, R. Wing. Reproductive Endocrinology Associates of Charlotte, Charlotte, NC. OBJECTIVE: The correlation of maternal age and aneuploidy has been well studied but the effect of paternal age on aneuploidy is still not clear. With the current trend of couples starting a family at a later age this becomes an important emerging issue. Here we examined the effect of paternal age on aneuploidy when controlling for maternal age. DESIGN: Retrospective, single clinic study. MATERIALS AND METHODS: Patients undergoing IVF with comprehensive chromosome screening at the blastocyst stage were included in the study. Patients were divided into four groups based upon paternal and maternal age: maternal age <35 years and paternal age <40 years (group 1), maternal age <35 years and paternal age R40 years (group 2), maternal age R35 years and paternal age <40 years (group 3), and maternal age R35 years and paternal age R40 years (group 4). RESULTS: A total of 43, 16, 55, and 55 patients were included in groups 1, 2, 3, and 4, respectively. Average paternal age between group 1 and group 2 was significant, 34.0
2.7 years and 39.8
5.0 years, receptively (P<0.0001). Average maternal age between group 1 and group 2 was not significant, 31.6
2.1 years and 31.3
1.6 years, respectively (P¼0.5782). Blastocyst aneuploidy rates were not significantly higher in group 1 compared to group 2, 107/172 (62.2%) and 50/77 (64.9%), respectively (P¼0.7873). Average paternal age between group 3 and 4 was significant, 36.8
2.5 years and 45.3
5.9 years, respectively (P<0.0001). Average maternal age between group 3 and 4 was not significant, 38.2
2.2 years and 38.9
1.3 years, respectively (P¼0.0536). Blastocyst aneuploidy rates were significantly higher in group 3 compared to group 4, 93/196 (47.5%) and 70/202 (34.6%), respectively (P¼0.0127). CONCLUSION: Our results indicate that paternal age has little to no influence in a younger maternal population (<35 years). However, there is a significant increase in blastocyst aneuploidy rates in an older maternal population (>¼35 years) when the male is >¼40 years compared to younger men (<40 years old). This may indicate that oocytes from older women cannot compensate for the paternal contribution to aneuploidy at the same rate as oocytes from young women. Supported by: Reproductive Endocrinology Associates of Charlotte.

P-651 Wednesday, October 22, 2014 INVO & ICSI: A PIONEER IDEA AND A REAL ALTERNATIVE FOR ART. E. Lucena, H. Moreno, O. Lombana, A. Moran, L. Coral, C. Esteban. Reproductive Medicine, CECOLFES (Colombian Fertility and Sterility Center), Bogota, Colombia. OBJECTIVE: Intravaginal culture of oocytes (INVO) is an assisted reproductive technique (ART) that allows oocyte fertilization and early embryo development inside of air free, gas-permeable (CO2 and O2) plastic device

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ASRM Abstracts

called INVOcellTM. This study reports an application of INVO procedure for patients displaying male factor. After intracitoplasmatic sperm injection (ICSI) procedure, microinjected oocytes are placed into INVO device to be incubated inside of vaginal cavity. A total of 172 of cycles of couples showing male factor as cause of infertility were included in the INVO – ICSI protocol, from January to December 2012 in CECOLFES as a novel optional treatment in ART, showing similar results compared to classical ICSI procedures. DESIGN: Mild ovarian stimulation, ICSI procedure, INVO technique, embryo transfer (ET), and detection of b hCG hormone after fourteen days of ET. MATERIALS AND METHODS: In the present study, a total of 172 cycles were undergone to INVO-ICSI protocol after giving consent. Natural cycle or mild controlled ovarian stimulation with clomiphene citrate (OMIFINÒ) and hMG (MerionalÒ) were used. An average of 4.7 microinjected oocytes per patient were placed in the INVOcellTM containing G2 PLUSÒ of VitroLife stabilized at 37 grades centigrade. Seminal samples were collected one hour before oocyte retrieval and enabled using density gradients protocol for sperm capacitation. ICSI was performed on each oocyte and all microinjected oocytes were placed into the INVO device. INVO was then inserted in the vaginal cavity during 72 hours for incubation and at that time it was removed to recover the embryos for immediate uterine embryo transfer. Detection of serum b hCG was performed fourteen days later. RESULTS: A total of 172 cycles were performed. On average 6.5 oocytes per cycle were retrieved, a mean of 4.7 microinjected oocytes were placed into INVOcell device, and a mean number of transferred embryos per cycle was 2.0. The cleavage rate obtained after the INVO culture was 53.1%. A total number of 65 clinical pregnancies (59 single pregnancies and 6 multiple pregnancies) were achieved corresponding to 37.9 % of pregnancy rate per cycle and to 40.3% of pregnancy rate per transfer. CONCLUSION: This study shows for the first time that INVO-ICSI protocol can be an effective and viable alternative treatment option in ART to achieve pregnancy after ICSI since the severity of male factors is 68% with comparable results to those reported using classical ICSI. Supported by: Colombian fertility and sterilitiy center - CECOLFES.

P-652 Wednesday, October 22, 2014 IVF OUTCOME IN AZOOSPERMIC CANCER SURVIVORS. S. Dar,a J. Levron,a J. Haas,a R. Machtinger,a A. Kedem,a I. Gat,a I. Madgar,b R. Orvieto,a G. Raviv.b aIVF Unit, Chaim Sheba Medical Center, Ramat Gan, Israel; bUrology, Chaim Sheba Medical Center, Ramat Gan, Israel. OBJECTIVE: The number of cancer survivors is increasing constantly and an average of 15-30% of them remain sterile in the long term As such, concerns about fertility and family planning are more relevant than ever. Successful pregnancies have been reported with IVF/ICSI utilizing testicular sperm. We therefore aim to evaluate the IVF outcome in azoospermic cancer survivors, who underwent testicular sperm extraction (TESE) and IVF/ICSI in our tertiary university-affiliated IVF unit. DESIGN: A retrospective cohort study. MATERIALS AND METHODS: All consecutive azoospermic cancer survivors who underwent TESE consisting of multiple random biopsies and IVF/ICSI, between 1996 to 2011were evaluated. ICSI procedure was performed using sperm retrieved. Fertilization was confirmed by the presence of two pronuclei (PN) on the day after ICSI. Embryo transfer (ET) was done on day 2 or 3. Clinical pregnancy was defined as the presence of an intrauterine gestational sac with embryonic pole diagnosed by ultrasonography. Demographic information, pretreatment hormones levels and TESE and ICSI outcomes were analyzed. RESULTS: During the study period, 31 cancer survivors underwent 39 TESE combined with IVF/ICSI cycles. The mean patients’ age and serum FSH level were 34.0
7.0 years and 18.8
10.4 IU/L, respectively. The average left and right testicular volumes were 17.2
4.2 ml and 13.4
8.8 ml, respectively. The mean time from chemotherapy to TESE was 8.2
2.6 years. Sperm was retrieved on the day of oocyte retrieval (fresh cycle). Sperm was successfully retrieved in 11 out of 31 patients (35.5%) on initial TESE, with an overall sperm retrieval rate of 38.4% (15 of 39). The average number of retrieved oocytes was 14.0
4.0 per cycle, with clinical pregnancy and live birth rates per successful TESE of 60% (9 of 15) and 53.3% (8 of 15), respectively. Age, serum FSH, testicular volume and time from chemotherapy to TESE were not significantly different between patients with successful TESE to those without successful TESE. Non Hodgkin lymphoma 1 0f 3, leukemia 0 of 3, solid tumors 4 of 11, Hodgkin lymphoma 4 of 11 and Seminoma

Vol. 102, No. 3, Supplement, September 2014