Neri, Queenie V BSc; Takeuchi, Takumi MD, PhD; Kang, Hey Joo MD; Lin, Kathleen MD; Wang, Alfred BA; Palermo, Gianpiero D MD, PhD Author Information

Antisperm antibodies (ASA) affect sperm transport and ultimately fertilization. ART techniques such as IUI and IVF have been able to alleviate this problem, however, specific classes of antibodies or certain combinations of these, can interfere with zona pellucida penetration and oolemma fusion when located on the sperm head. For these cases, the use of ICSI is required. We plan to validate various clinical indications to justify ASA testing. In addition, the effect of ASA on clinical parameters such as fertilization, cleavage, and pregnancy outcomes were investigated. In a case control fashion, we assessed the usefulness of ASA screening by comparing ICSI outcome. A total of 100 couples screened for ASA were treated in 167 ICSI cycles, from January 2000 to December 2004. Antibody testing was performed by direct immunobead assay for IgG, A, and M. Up to 100 motile spermatozoa was scored for immunobead binding. According to the WHO criteria, positive samples were those with the presence of ≥ 50% antibodies of all classes (IgG, A, or M). In addition, to antibody titers, their location on the sperm cell (head, midpiece, tail) was also recorded. Couples were then divided into three groups whether they had positive (≥ 50%), intermediate (< 50%), or negative (control) ASA. Data collected included, age, indication for ASA testing, oocytes retrieved, oocytes injected, fertilization, embryo quality, clinical pregnancy, and delivery outcomes. The detection rate among those clinically suspected to have ASA was 13.0% (13/100). History of testicular surgery (37.5%), history of positive ASA elsewhere (50.0%), agglutination on semen analysis (10.6%), and poor post-coital test (13.3%) were the main indications for ASA screening. Surprisingly, none of the men who had undergone vasectomy reversals tested positive for ASA. The large majority of the ASA positivity was on the sperm head, tail, or both. In addition, regarding the antibody class the association between IgG and IgA (69%) was the most recurrent. There was an inverse correlation between antibody titer and fertilization with the ≥ 50% positivity that had 68.1% fertilization, while the < 50% group had 84.4%, and the controls 76.2%. Embryo cleavage, clinical pregnancies, and deliveries were similar among the three groups. In spite of a mild effect on fertilization rate, ASA did not affect pregnancy outcome. Among the indications, history of ASA and testicular surgery had the highest validation in predicting antibody presence. There was no additional effect by the localization of the antibodies on the spermatozoa or antibody class. Considering that infertile couples are often steered to the ICSI procedure for other indications, antibody testing itself may have a marginal usefulness.

In a large IVF center, there is always a complication in building a user-friendly information system, aiming at elimination of human error and minimizing data entry effort required for the end-user, who might have limited knowledge of computers. Yet the sytem should be able to fulfill the Center's daily data retrieval demand, consistently and concurrently produce medical documents, perform simple statistics, and generate reports for annual health department surveys. Moreover, since the implementation of the HIPAA regulation in the US, the privacy of patients' health information is paramount. A well-designed information system can act as a first line to ban unauthorized information access. We developed a user-friendly database through which a reproductive laboratory can store data real-time, provide specific information to each user, and quickly browse information. Here, we compared the efficiency and accuracy of such a customized user-friendly database with that of a conventional hard copy and spreadsheet system. Five tables containing andrological information and patient demographics were linked togher with an identifier. End-users were trained to enter and perform queries in the database. A computer designated in a local area network acted as a server containing the user-friendly database. All users attended a one hour training class. Tests for both conventional (spreadsheet) and study databases were run for all recurrent clinical and research scenarios. The user-friendly database included 5 tables: Patient Identification (PI), Semen Analysis (SA), Intrauterine Insemination (IUI), Cryopreservation (Cryo), and Cryostorage of Surgical Specimens (CSS). From September 2002 to Arpil 2005, we recorded 4,418 demographic data in the PI table, 3,418 SA, 4,969 IUI, 807 Cryo, and 277 CSS cycles. The information for each table can be entered directly at the laboratory bench as soon as it becomes available. A search engine was developed according to the needs of the users. At the end of each analysis, the screen print-out generated a hard copy for laboratory filing and a copy for patient charts.Regarding security, only specific personnel were granted privileges where a password had to be entered prior to gaining access or entering records into the study database. The privileges for each user are tailored to the individual's needs and role in the Center. Therefore, the possibility of accidentally altering results or leakage of patients' information is significantly reduced. Although, it took approximately 6 months to develop, this database system reduced the time required for technicians to transcribe, classify, photocopy, and enter a specific record of over 90%. The conventional paper/spreadsheet system is often prone to many inconsistencies. The customized user-friendly database on the other hand, allows a single entry, provides secure data storage, and is only a click away. Moreover, the different levels of access bring greater security. Finally, because it is user-friendly, the screen is identical to the original paper reports, the adoption of a user-friendly database is simple and easy for both medical and technical staff alike.

Depending on different stimulation protocols and varying patient response, about 70–80% of the oocytes are metaphase II at egg retrieval. When the contribution of the immature oocytes is higher, particularly when over 50%, this is cause of great distress to physicians and patients. In standard in vitro fertilization, the proportion of mature oocytes can be determined only on the day of fertilization assessment, whereas in the case of ICSI this becomes obvious immediately after cumulus removal, allowing insemination of only mature oocytes. In the present study, we aimed to assess the impact of a high proportion of immature oocytes present at retrieval on ICSI outcome. Patients who underwent the ICSI procedure were classified retrospectively according to the proportion of immature oocytes at the time of retrieval. Term outcomes were fertilization, implantation, and pregnancies. Couples treated by ICSI from September 1993 to December 2001 were included in this study. In order to exclude confounding factors related to maternal age or severe male factor, only women ≤35 years of age and only ejaculated spermatozoa were used. Ovarian stimulation was performed with a GnRH-agonist/antagonist and gonadotropins, with oocytes retrieved approximately 36 hours after hCG administration. ICSI was then performed only on MII oocytes in a standard fashion. Fertilization was assessed 16–18 hours after insemination and good quality embryos were transferred to the patients either on day 3 or day 5. Patients were stratified into three groups: with 100% maturation (complete), those with 50% of the oocytes matured (partial), and those with only 30% mature (minimal). A total of 2,368 ICSI cycles using ejaculated spermatozoa were assessed, with a similar maternal age in all groups and an overall average maternal age of 32.1 ± 3 yrs (range 18–35 yrs). The number of cycles for the complete maturation group was 388 (16.4%), for the partial maturation was 116 (4.9%), and for the minimal maturation was 31 (1.3%). The total number of oocytes retrieved was 3,274, 624, and 87, respectively, while the average number of oocytes injected was 8.4, 5.5, and 3.9. The fertilization rates and patterns were not different among the three cohorts, being 73.5, 69.7, and 76.9%, respectively, and the implantation rate was directly related to number of mature oocytes (28.9, 31.8, and 23.8%). Proportional differences were found in the pregnancy (46.4, 50.0, and 29.0%; P < 0.01) and in delivery rates (44.3, 44.8, and 25.8%; P < 0.01). Thus, while the outcomes for the complete and partial maturation gorups were comparable, the minimal maturation group had a lower rate of success. Within the cohort of oocytes retrieved the proportion of those at the MII stage did not affect fertilization outcome as long as at least half of them were mature. Only when ∼33% of mature oocytes was present was when the ICSI outcome compromised. Although the lower number of mature oocytes did not impair quality of any embryos eventually produced as judged by the rates of implantation, this still impairs pregnancy outcome because of a lower number of embryos transferred.

AIM:Intracytoplasmic sperm injection (ICSI) is now the preferred technique for treatment of male factor infertility and many children have been born worldwide. However, concerns about the risk of transmitting genetic defects and development of ICSI children have been raised. We report clinical outcome of ICSI in Cornell University and results of screening for genetic defects in ICSI parents and their children.METHODS:Pregnancy and obstetrical outcomes as well as congenital malformations were analyzed. Chromosomal karyotyping and Yq deletion assessments were performed on blood samples. In addition, medical and developmental outcome were assessed in 3 and 5 year old ICSI children.RESULTS:We have performed 8 575 ICSI cycles with ejaculated spermatozoa that resulted in a 75.4% fertilization and a 42.8% clinical pregnancy rates while for surgically retrieved specimen resulted in 66%, 48.8% respectively. The incidence of Y deletion was within the expected range for an infertile population, with identical deletions transmitted to the male offspring. There were no differences in cognitive, motor and behavioral development observed between ICSI children and these conceived naturally.CONCLUSION:The large majority of infertile men were treatable by ICSI, which resulted in the transmission of a specific abnormality but did not enhance the incidence of de novo deletions. There is no indication that ICSI children develop more congenital defects or express a lower psychomotor development that children conceived naturally. Nonetheless, genetic screening and counseling of couples undergoing ICSI would seem to be appropriate.

The MetaCyc database (see URL http://MetaCyc.org ) is a collection of metabolic pathways and enzymes from a wide variety of organisms, primarily microorganisms and plants. The goal of MetaCyc is to contain a representative sample of each experimentally elucidated pathway, and thereby to catalog the universe of metabolism. MetaCyc also describes reactions, chemical compounds and genes. Many of the pathways and enzymes in MetaCyc contain extensive information, including comments and literature citations. SRI’s Pathway Tools software supports querying, visualization and curation of MetaCyc. With its wide breadth and depth of metabolic information, MetaCyc is a valuable resource for a variety of applications. MetaCyc is the reference database of pathways and enzymes that is used in conjunction with SRI’s metabolic pathway prediction program to create Pathway/Genome Databases that can be augmented with curation from the scientific literature and published on the world wide web. MetaCyc also serves as a readily accessible comprehensive resource on microbial and plant pathways for genome analysis, basic research, education, metabolic engineering and systems biology. In the past 2 years the data content and the Pathway Tools software used to query, visualize and edit MetaCyc have been expanded significantly. These enhancements are described in this paper.

Serious doubts have been raised about development of children born after assisted reproductive technologies (ART) such as IVF and ICSI. These qualms are more specifically related to the possibility of gene defects, whether linked to imprinting patterns or gene mutations. Well-characterized imprinting diseases in humans include Angelman and Beckwith-Wiedemann syndromes. These disorders are the result of defects in DNA methylation, interfering with gene expression and leading to growth pattern distortion, mental retardation, and slow language development. These putative problems have been attributed to the in vitro culture environment or the fertilization method utilized. The aim of this study to was to assess whether extended in vitro of embryo culture from them has any affect on the overall well-being of the ART children. Children born following standard in vitro fertilization or ICSI were recruited to participate in a follow-up questionnaire study. End term results assessed were ART outcome as a function of day 3 versus day 5 embryo transfer. The patients recruited underwent ICSI and standard IVF from June 1996 to July 2000. ART criteria included the number of MII oocytes, fertilization rate, number and age of embryos, as well as pregnancy outcome. Neonatal measures such as birthweight, gestational age, and apgar scores also were recorded. Children were stratified according to whether embryos were transferred at either day 3 or at blastocyst stage. Consenting parents of ART children of 3 yrs ± 6 mos (IRB # 0399–613) completed the AgessStages Questionnaire (ASQ), Social Skills Rating System (SSRS), and Parenting Stress Index (PSI). According to the score, the child was classified as developing typically, or as needing further evaluation (i.e. "at risk"). A total of 5,842 patients had a transfer on day 3 and 299 on day 5. The mean maternal ages were comparable (36.1 ± 5 vs 35.1 ± 4, respectively) and the fertilization rate for day 3 was 74.7%, and was 80.0% for day 5. No differences were found between the rate of abnormal 1PN or 3PN formation in either group. However, as expected the number of embryos transferred on day 3 (3.3) was significantly higher (P < 0.01) than the 2.9 transferred on day 5. The pregnancy (45.4 vs 56.5) and delivery (39.2 vs 50.8) rates between the two groups were similar. The birthweight was 3326 ± 573 and 3505 ± 456, respectively and the gestational age as well as apgar scores were comparable. In regard to development, a total of 491 of the day 3 group completed at least one questionnaire and 17 did so for the day 5 group. Moreover, among those who participated, there was a similar rate of multiple gestation between the two group (44.4 vs 36.4%, respectively). The ASQ revealed no significant difference in the incidence of 'at risk' children in the day 3 (10.0%) compared to the day 5 group (12.5%), nor were differences evident in their social-behavior. The stress level and parent-child interactions were within the norm for all groups. Extended in vitro culture significantly lessens the number of embryos being transferred to the patient, so reducing the occurrence of multiple gestation. However, it appears that the in vitro culture systems employed do not compromise the development of ART children. Though more extensive monitoring seems justified, these results help to ease concerns about the possible impact of extended culture on gene expression in the embryo.

Since the successful introduction of sequential media for extended culture the replacement of embryos as blastocysts has become common practice in many IVF programs. Sequential media are distinguished by a well-characterized supplementation of vitamins and amino acids in contrast to undefined serum proteins present in conventional culture media. When a higher concentration of serum is added to the insemination medium, possibly altering oolemma structure, an improved cell survival after ICSI has been reported. Furthermore, it has been demonstrated where ICSI fertilization is abnormal e.g. in the case of tri-pronuclear digyny this is associated with enhanced oolemma fragility. Here we aimed to identify any eventual effect of different culture media on ICSI fertilization characteristics. We analyzed embryological parameters of patients undergoing ICSI, retrospectively. Term outcomes included normal and abnormal fertilization, number of embryos replaced, and pregnancy characteristics. ICSI cycles using ejaculated spermatozoa (n = 4638) with concentration ≥ 20 × 106/ml from September 1993 to September 2003 were included. MII Oocytes retrieved approximately 36 hours after hCG, following controlled ovarian stimulation, were subjected to ICSI in a standard fashion. Fertilization was assessed 16–18 hours later with the presence and characteristics of pronuclei recorded. Embryos were then transferred either on day 3 or 5 according to availability and quality. Clinical pregnancy rates and the gestational order were assessed by the presence and number of fetal heartbeats. A total of 4,638 ICSI cycles were stratified by the culture media employed. The conventional media group included 1,921 ICSI cycles in which 19,670 oocytes were retrieved. 15,307 MII oocytes were injected and 14,460 survived, resulting in the formation of 11,857 oocytes with 2PN, 451 with 1PN, and 643 with 3PN. There were 784 clinical pregnancies, 443 singletons, 238 twins, and 103 high-order multiples. The sequential culture media group included 2,717 ICSI cycles in which 22,440 MII oocytes were injected and 20,872 survived, resulting in the formation of 17,269 with 2PN, 483 with 1PN, and 642 with 3PN. A total of 1,048 clinical pregnancies produced 668 singletons, 278 twins, and 102 high-order gestations. The use of conventional media was correlated with a higher oocyte survival (P < 0.0001), but also with an increase of oocytes with 3PN (P < 0.0001) and 1PN (P < 0.0001), with the incidence of 2PNs being similar between the two media. Switching to the sequential culture media by increasing blastocyst transfer rate brought an increase in singletons (P < 0.01) and a reduction in multiple gestations (P < 0.05). Sequential media were associated with a decreased oocyte cell-survival rate perhaps reflected in effects exerted at the level of the plasmalemma. Extended culture conditions appear to favor both a decrease in fertilization abnormalities and a reduction in gestational order.

Objective: The removal of cumulus cells for ICSI allows the observation of oocyte nuclear maturity. Oocytes retrieved at the germinal vesicle (GV) stage require overnight culture to reach metaphase II (MII), but only about 50% of those extrude a polar body, and once fertilized, display poor embryonic cleavage. Immature oocytes at metaphase I (MI) are conventionally considered equivalent to the MII oocyte in terms of performance, since they reach maturation within a few hours from the retrieval, and are often used for replacement. In this study, we analyzed the frequency of incomplete maturation at the time of oocyte retrieval for ICSI cycles. Design: After cumulus cell removal, the nuclear maturity of the oocytes was assessed, and for those at MI stage, maturation timing, fertilization efficiency, and embryonic cleavage were evaluated. Materials and Methods: The evaluations involved ICSI cycles in which at least one in-vitro matured MI oocyte was injected. Oocyte retrieval was performed approximately 35–36 hrs following hCG administration after controlled ovarian stimulation with GnRH agonist and gonadotrophins. The oocytes were studied immediately after cumulus cell removal, for extrusion of the first PB as an indication of the MII stage. MI oocytes were designated as those that had undergone GV breakdown, had mature cytoplasm, and had not yet extruded a PB, as distinct from those displaying a GV. Thus, the study group included oocytes identified as MI's at time of cumulus cell removal, and those that reached the MII stage up to 42 hrs after hCG administration. Oocytes were injected by ICSI in a standard fashion and cultured individually. Fertilization characteristics based on a second PB and pronucleus development were evaluated 16–18 hours after ICSI, and embryonic cleavage was evaluated at 24 hr intervals until the day of the transfer. Oocytes at MII stage at time of cumulus cell removal served as controls. Results: Of a total of 907 ICSI cycles with ejaculated spermatozoa, 152 (16.7%) had at least one MI oocyte. In this female population of 36.7 ± 4 years, the distribution of oocyte nuclear maturation was 66.9% at MII, 18.7% at MI and 12.5% at GV stage. During a brief post-retrieval culture period of up to 4 hours, 55.2% of 359 of MI oocytes extruded a first PB. The fertilization rate of 44% (89/198) was much lower than the 74.6% rate in 1286 MII oocytes (P < 0.0001). Their survival and rates of abnormal fertilization were similar, as was embryonic cleavage (36.7% of 960 for MII, and 34.8% of 89 for MI). Because they were transferred together with embryos derived from MII oocytes, it was not possible to obtain the pregnancy rate for embryos developing from MI stage oocytes. Conclusion: Of the cycles assessed, 16.7% had at least one MI oocyte at the time of cumulus cell removal that nevertheless matured within a four hours period. Although the in-vitro matured MI oocytes exhibited a significantly lower fertilization rate than those at MII, the cleavage patterns of those fertilized were similar. Thus, although fertilized at a lower rate, MI oocytes can be successfully injected, they do not reveal a higher incidence of fertilization abnormality and can be included in the cohort being evaluated for embryo cleavage and eventual transfer. However, caution should be taken in including embryos from these recently matured oocytes into the cohort of embryos selected for transfer because in vitro development of blastocyst and implantation capability of these oocytes need to be further elucidated.

Objective: Some of the protocols used in ART have revised concerns in regard to the appearance of uncommon diseases, e.g. those related to gene imprinting defects. ICSI often requires the use of chymotrypsin, or pentoxifylline, or both to isolate a few spermatozoa from in severely oligo/azoospermic men, but the use of these compounds during sperm preparation for ICSI has been a source of concern for neonatal outcome and child development. Therefore, we have analyzed embryo development, implantation, neonatal outcome, and subsequent development of ICSI offspring conceived with spermatozoa that have been prepared using these compounds.Design: Prospective follow-up study on the neonatal outcome, and psychological, cognitive, and behavioral profiles of ICSI children. According to specimen characteristics, spermatozoa for ICSI were treated with chymotrypsin, pentoxifylline, with a combination of both, or none.Materials and Methods: Epididymal and testicular specimens were obtained microsurgically, and ejaculated samples were collected by masturbation, following which chymotrypsin (200 to 500 IU) was used to promote liquefaction. Spermatozoa with poor motility were treated by 3 mM pentoxifylline. After ICSI, the neonatal outcomes later measured included birthweight, gestational age, and apgar scores. Congenital malformations were considered as major when these required surgical intervention or hampered function, and as minor in the absence of these problems. Consenting parents of children of 3 yrs ± 6 mos (IRB # 0399–613) and 5 yrs ± 6 mos (IRB # 0299–581) completed the Ages & Stages Questionnaire (ASQ), Social Skills Rating System (SSRS), and Parenting Stress Index (PSI). According to the child’s score, they were classified as developing typically, or as needing further evaluation (i.e. ‘at risk’).Results: A total of 5,294 ICSI cycles yielded 16,627 embryos, of which 3,670 (22.1%) developed a positive fetal heartbeat, and 2,901 newborns were delivered. The minor malformation rate was 0.7% (n = 21) and the major was 1.1% (n = 32). Among 2,901 babies, 667 were conceived with chymotrypsin-treated sperm, 151 with pentoxifylline-treated sperm, 42 with chymotrypsin and pentoxifylline, and 2,041 without treatment (control). Whichever the treatment, and particularly a combination of these, had some negative effect on fertilization (P < 0.01), there were no notable differences subsequently in the implantation rates, neonatal outcome, or malformations. In regard to development, 27.2% of 716 ICSI families completed at least one questionnaire and among these, 233 were born from 195 deliveries. The ASQ revealed no significant difference in the incidence of ’at risk’ children in the chymotrypsin group (6.0%) compared to the control (3.5%), nor were differences evident in their social and behavioral character (4% chymotrypsin vs 8% control). The stress level and parent-child interaction were within the norm for all groups.Conclusion: Chymotrypsin, pentoxifylline, or a combination of the two used to prepare spermatozoa of severe oligo/azoospermic men tended to reduce fertilization rates, but not implantation rates, and there were no significant differences for the ICSI children conceived with these sperm compared to naturally. Though further monitoring may be justified, these results help to alleviate concerns that the treatments to which gametes and embryos are subjected have no consequence for the wellbeing of ART children. Objective: Some of the protocols used in ART have revised concerns in regard to the appearance of uncommon diseases, e.g. those related to gene imprinting defects. ICSI often requires the use of chymotrypsin, or pentoxifylline, or both to isolate a few spermatozoa from in severely oligo/azoospermic men, but the use of these compounds during sperm preparation for ICSI has been a source of concern for neonatal outcome and child development. Therefore, we have analyzed embryo development, implantation, neonatal outcome, and subsequent development of ICSI offspring conceived with spermatozoa that have been prepared using these compounds. Design: Prospective follow-up study on the neonatal outcome, and psychological, cognitive, and behavioral profiles of ICSI children. According to specimen characteristics, spermatozoa for ICSI were treated with chymotrypsin, pentoxifylline, with a combination of both, or none. Materials and Methods: Epididymal and testicular specimens were obtained microsurgically, and ejaculated samples were collected by masturbation, following which chymotrypsin (200 to 500 IU) was used to promote liquefaction. Spermatozoa with poor motility were treated by 3 mM pentoxifylline. After ICSI, the neonatal outcomes later measured included birthweight, gestational age, and apgar scores. Congenital malformations were considered as major when these required surgical intervention or hampered function, and as minor in the absence of these problems. Consenting parents of children of 3 yrs ± 6 mos (IRB # 0399–613) and 5 yrs ± 6 mos (IRB # 0299–581) completed the Ages & Stages Questionnaire (ASQ), Social Skills Rating System (SSRS), and Parenting Stress Index (PSI). According to the child’s score, they were classified as developing typically, or as needing further evaluation (i.e. ‘at risk’). Results: A total of 5,294 ICSI cycles yielded 16,627 embryos, of which 3,670 (22.1%) developed a positive fetal heartbeat, and 2,901 newborns were delivered. The minor malformation rate was 0.7% (n = 21) and the major was 1.1% (n = 32). Among 2,901 babies, 667 were conceived with chymotrypsin-treated sperm, 151 with pentoxifylline-treated sperm, 42 with chymotrypsin and pentoxifylline, and 2,041 without treatment (control). Whichever the treatment, and particularly a combination of these, had some negative effect on fertilization (P < 0.01), there were no notable differences subsequently in the implantation rates, neonatal outcome, or malformations. In regard to development, 27.2% of 716 ICSI families completed at least one questionnaire and among these, 233 were born from 195 deliveries. The ASQ revealed no significant difference in the incidence of ’at risk’ children in the chymotrypsin group (6.0%) compared to the control (3.5%), nor were differences evident in their social and behavioral character (4% chymotrypsin vs 8% control). The stress level and parent-child interaction were within the norm for all groups. Conclusion: Chymotrypsin, pentoxifylline, or a combination of the two used to prepare spermatozoa of severe oligo/azoospermic men tended to reduce fertilization rates, but not implantation rates, and there were no significant differences for the ICSI children conceived with these sperm compared to naturally. Though further monitoring may be justified, these results help to alleviate concerns that the treatments to which gametes and embryos are subjected have no consequence for the wellbeing of ART children.

Objective: Notwithstanding the large number of babies born from the ICSI procedure worldwide, concerns still exist because of the use of suboptimal spermatozoa, known to be associated with a higher incidence of genomic abnormalities. Follow-up of the ICSI children during their development is highly recommended. Since assessment of psychological and physical development is costly and time consuming, the use of parent-administered questionnaires has been proposed as way of monitoring the development of ICSI offspring. Design: Prospective survey using a home-administered questionnaire to compare the psychological and physical development of 3 year old children conceived by ICSI and standard IVF, respectively. Materials/Methods: Consenting parents (IRB # 0399–613) of 3 year old (±6 months) children completed the Ages & Stages Questionnaires (ASQ), a series of developmental questionnaires. The ASQ is an illustrated 30-item questionnaire for assessing child development (from birth to 5 years of age) in their natural environment. Five key developmental areas—communication, gross motor, fine motor, problem solving, and personal-social—were evaluated in addition to a section addressing specific parental concerns. Questionnaires were ranked according to the child's score, as 'typical' development or 'at risk' therefore needing further evaluation. Results: Of 450 ICSI families invited, only 20.0% (n = 89) responded and only 11.9% (36/302) of the IVF families completed the questionnaires. Based on the ASQ screening cut-off points, 86.5% (77/89) of the ICSI children displayed normal development, comparable to the 88.9% (32/36) of IVF children. A total of 12 (13.5%) ICSI children were considered 'at risk', five coming from multiple pregnancies, while in the IVF group four (11.1%) children were found to be 'at risk', with two being from a high order gestation. There were no differences between boys and girls among the five developmental areas. Conclusions: This study indicates that the ASQ provides a cost- and time-effective method through which to monitor the development of children resulting from assisted reproductive technology, particularly for centers where a pediatric and psychological follow-up would be inconvenient. The 3 year old children assessed in the ICSI and IVF groups, are developing well without significant delays in the cognitive abilities, socio-emotional development, and motor skills. The low response rate may be attributable to the confidence that parents have in the normality of their ART children. Because of the relatively low response rate, definitive conclusions cannot be drawn as yet. Supported by: This study was partially supported by an unrestricted grant from Organon Inc., USA.

Objective: To assess the genetic status of ICSI families comprised of mother, father, and their offspring. ICSI is now the preferred technique for treatment of male factor infertility and several thousands of children have been born worldwide. However, since male subfertility has been associated with a higher incidence of genomic defects, ranging from numerical chromosomal abnormalities to Yq microdeletions, concerns have been raised as to the risk of transmitting genetic defects to the offspring. Screening for such defects can provide invaluable information for appropriate counseling prior to ICSI treatment. Design: Prospective follow-up of ICSI couples and their offspring for chromosomal abnormalities and incidence of Y microdeletions. Materials/Methods: A total of 96 consenting families: 95 mothers (38 ± 4 yrs), 66 fathers (44 ± 6 yrs), and 91 children (5 ± 0.5 yrs) provided at least one of the following - blood, semen, and/or cheek cells (IRB # 0299–581). Peripheral lymphocytes were assessed for numerical chromosomal abnormalities by an external laboratory through standard G-banded karyotyping. Cheek cells were collected by vigorous scraping of the cheek mucosa at least 10 times with a specially designed cotton brush. DNA was isolated from blood using the Wizard Genomic DNA Purification Kit (Promega), from semen pre-treated with DTT and Proteinase K followed by protein precipitation, and from cheek cells by the Phenol/Chloroform method or the silica coated magnetic particles method. In the AZF region of the Y chromosome, a total of 22 Sequence Tagged Sites (STSs) were analyzed by multiplex PCR. The products of PCR were electrophoresed in TBE solution on 4% NuSieve 3:1 agarose gel, and these were analyzed under UV light. Results: A total of 237 blood samples were subjected to chromosomal analysis. Cytogenetic analyses were performed on mothers (n = 93), fathers (n = 65), daughters (n = 47) and sons (n = 35). All the adults and children had normal karyotypes except for one child with a mosaic Klinefelter, another with a complete Klinefelter form, and a third with an XYY syndrome (3.6%, 3/82). The father of the child with the mosaic Klinefelter, the only one willing to provide a blood sample, had a normal male karyotype. A total of 98 blood samples, 101 cheek cell specimens, and 26 semen samples were analyzed for Y chromosome microdeletions. All DNA extraction and PCR amplifications were successful. The mean sperm concentration was 39.0 ± 53 × 106/ml for ejaculated samples (n = 40) and 0.9 ± 1.1 × 106/ml for those retrieved surgically (n = 2). Microdeletions in the AZFb, AZFd, and AZFc regions were detected in three fathers (5.1%, 3/59), two of which had a daughter and one a son with an identical Yq deletion. Conclusions: In a follow-up study to evaluate the development of ICSI offspring, genetic screening revealed that, compared to the general population, these children had a higher incidence of gonosomal abnormalities (5.1%), possibly arising de novo. Sex chromosomal abnormalities are increased in offspring of infertile men and the incidence of Yq deletion was within the expected range for azoo-/oligospermic population. Therefore, genetic screening of couples undergoing infertility treatment and of eventual offspring is still highly recommended, particularly for detection of de novo chromosomal abnormalities and for Yq microdeletions. Supported by: Cornell Institute for Reproductive Medicine, New York, NY.