Endocervical lavage technique for embryo transfer improves pregnancy outcome in assisted reproductive techniques

Balaban, Alper Mumcu, Cengiz Alatas, Bulent Urman. American Hosp of Istanbul, Istanbul, Turkey. Objective: Early incubation of the ejaculate with medium have several theoretic advantages including a decrease in spermatozoa containing antisperm-antibodies, removal of bacteria and detritus, a protective effect against free oxygen radicals and sperm DNA protection. It has been postulated that early dilution of ejaculate may improve fertilization due to aforementioned beneficial effects. The aim of this study was to investigate if early incubation of semen samples into media enhances fertilization rates in unselected group of patients undergoing ICSI. Design: Prospective-randomized study. Materials and Methods: One hundred and nine patients who underwent ICSI were evaluated. There were no selection criteria regarding the indication for ICSI. All couples were randomly divided into two groups. In group I semen was collected into sterile dry pots and in group II semen was collected into 5 ml of bicarbonate buffered medium containing human serum albumin (IVF-100, Vitrolife), mixed gently and incubated for 15-20 minutes at 37 C. Subsequently in both groups semen samples were washed with Pure Sperm gradients of 90, 70 and 50 at 250-500 gr for 5 minutes. Afterward samples were washed for a second time with 10 ml of IVF-100 at 500-750 g for 10 minutes. All native semen samples were evaluated manually according to WHO criteria (WHO, 1992) and morphology by Kruger strict criteria’s. Also evaluation was performed as above after preparation. After incubation for 16-18 hours oocytes were checked for presence of two pronucleus. Results: Indications for ICSI were unexplained infertility and oligoasthenoteratospermia. As shown in table I, no difference was observed regarding age, sperm count, morphology and motility in native semen samples, number of retrieved oocytes, fertilization and number of embryos transferred between two groups. Also there was no difference in semen parameters after preparation. Clinical pregnancy rates were 42.3% and 54.4% in group I and II, respectively. The difference was insignificant.

tocyst transfers. Of the 103 patients in the glass syringe group, 21 were frozen embryo transfers and 27 were blastocyst transfers. All others were Day 3 transfers. Average number of embryos transferred and the average age were not significantly different in the two groups. All patients who had rising beta hCG levels were considered pregnant. In addition, the difficulty of transfer and any debris, mucus, blood or tissue inside the inner catheter after the embryo transfer was noted. Results: Pregnancy rates were 50% in the plastic syringe group and 51.5% in the glass syringe group. There were three chemical pregnancies and all were in the glass syringe group. There was no significant difference in the number of embryos transferred and the average age of patients. There were eight patients with difficult transfers in the plastic group compared to nine patients in the glass group. One striking difference was that the inside of the transfer catheter was always cleaner (only five patients had some debris, mucus, tissue or blood inside the inner catheter) when glass syringe was used. When plastic syringe was used 29 patients had either debris, blood, mucus or tissue even though the physicians were careful to keep the plunger pushed after the transfer during the withdrawal of the catheter. However, 15 of these 29 patients with mucus, debris, blood or tissue in the catheter after transfer got pregnant. In addition, when glass syringe was used at least one embryo remained in the catheter after transfer and discovered during the flushing of the catheter in four patients. Only one patient had an embryo remaining after transfer when plastic syringe was used. Three of these five patients, when the embryo was retained and transferred, again got pregnant. Conclusion: Using the current embryo transfer technique using either all plastic or glass syringe, there appears to be no difference in the embryo transfer outcome. This is a surprising finding as we expected higher pregnancy rates in the glass syringe group, particularly when the catheters were cleaner after the transfer. Nevertheless, presence of debris, mucus, blood or tissue in the inner catheter after transfer does not appear to significantly decrease the conception. Significance of the slightly increased retention of embryos in the catheter after transfer is not clear. It could be due to tightness of the plunger in the glass syringe.

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Conclusion: Although there is a tendency to increase fertilization and pregnancy rates when semen sample is collected into medium, the difference was found to be insignificant. This may be due to small number of patients and larger studies are required to clarify the effect of early incubation of semen sample in medium on fertilization and pregnancy rates. P-27 Comparison of all plastic syringe (Henke-Sass) and glass syringe (Hamilton) for embryo transfer. Mahendran Mahadevan, Dean Moutos, Mahlon Maris, Michael Miller. Univ of Arkansas for Medical Science, Little Rock, AR. Objective: To compare the use of all plastic syringe with the glass syringe for embryo transfer. The volume of culture media aspirated can not be accurately controlled and the media containing the embryos are expelled with higher force during transfer with plastic compared to glass syringe. Design: Retrospective analysis of outcome data of alternate patients allocated to both groups. Materials and Methods: Alternate patients undergoing embryo transfers were randomly allocated to either plastic or glass syringe for aspiration of the embryos into the catheter for embryo transfer. Embryos were loaded in approximately 20 micro liters (15 to 25) of culture medium without any air bubbles. Embryos were loaded in the middle of the aspirated medium or slightly towards the tip of the catheter. Outcome of embryo transfers of the two embryologists and the two physicians have been consistently shown to be not significantly different in our program. Of the 102 patients in the plastic syringe group, 24 were frozen embryo transfers and 28 were blas-

FERTILITY & STERILITY威

Endocervical lavage technique for embryo transfer improves pregnancy outcome in assisted reproductive techniques. Gautam N. Allahbadia, Goral N. Gandhi, Kulvinder Kaur, Sonia Malik, Javaid Mugloo, Rubina Merchant. Rotunda-The Ctr For Human Reproduction, Mumbai, India; Rotunda-Virk Ctr For Human Reproduction, Jalandhar, India; Southend Rotunda Ctr For Human Reproduction, Delhi, India; Rotunda-Hygeia Ctr For Human Reproduction, Srinagar, India. Objective: To determine if the endocervical lavage with culture medium at the time of embryo transfer (ET) in patients undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) has any effect on pregnancy rates. Design: Retrospective, Multicentre, Comparative Study. Materials & Methods: All Embryo Transfers were done by the first author. From April 2001 to December 2001, all embryo transfers were done without using the endocervical lavage technique (n⫽73) and this was considered as the Control group. From January 2002 to March 2002, all transfers were done with an Edwards-Wallace™ ET catheter using the endocervical lavage technique (n⫽41) and this was the Study Group. Prior to the embryo transfer, a 2cc luer lock disposable syringe filled with the upgraded B2 INRA culture medium™ (CCD, France) is attached to the outer cannula of the Edwards-Wallace™ ET catheter. The cannula is gently advanced into the cervical os up to the internal os. Forceful squirts of 0.5 cc boluses of the culture medium are injected into the cervical os. Cervical mucus and culture medium can be seen escaping from around the cannula. After the Lavage, the embryologist loads the embryos into a new EdwardsWallace™ ET catheter and an embryo transfer is then done using the standard technique. All ETs were performed on Day 2 after egg retrieval. A clinical pregnancy was defined as the presence of an intrauterine gestational sac. Results: There was no significant difference between the Endocervical Lavage group and the Control group, respectively, in primary diagnosis, duration of infertility, endometrial thickness on day of transfer, grades of embryos transferred and the number of embryos transferred (5.19 3.05 versus 5.50 2.92). A clinical pregnancy (CP) rate of 31.71% was reported

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for the Endocervical Lavage group versus a 20.55% CP rate for the Control group. Conclusion: These data suggest that the Endocervical Lavage Technique for Embryo Transfer may enhance the likelihood of pregnancy. Removal of cervical mucus allows more effective delivery of embryos to the uterus. Microorganisms present in the cervical mucus are washed away and not introduced into the uterus during transfer. The Upgraded B2 INRA medium contains penicillin & streptomycin which may help to reduce the innoculum of micro-organisms into the uterus thus creating a favorable environment for implantation.

P-29 Prospective randomized comparison of an ultrasound-guided embryo transfer versus a blind catheter placement. Guillermo Marconi, Edgardo Young Jr., Martin Vilela, Ariel Bello, Edgardo Young Sr., Carlos Sueldo. Inst de Fertilidad, Buenos Aires, Argentina. Objective: It is generally accepted that an atraumatic embryo transfer is essential for successful implantation (Schoolcraft, 2002). Recently, we showed that soft catheters cause significantly less endometrial trauma than rigid or semirigid catheters (Marconi et al, 2003). Our objective is to evaluate the performance of an ultrasound guided echodense tip catheter vs. a commonly used semirigid catheter placed without ultrasound guidance in good prognosis IVF patients. Design: A prospective randomized study in a University-Affiliated IVF Unit. Materials and Methods: A total of 83 IVF patients were included in the study. They were all under 38 years of age and had a day 3 FSH under 12 IU/ml. Controlled ovarian hyperstimulation was done by using rFSH from either day 2 or 3 at 225 IU s.c. daily. The GNRH antagonist at 0.25 mg. s.c. daily was given when the largest follicule reached 14-15 mm in diameter. HCG at 10,000 IU was administered when the leading follicule was at least 18mm in diameter 35-36 hs prior to oocyte retrieval. Embryo transfer was performed on day 3 post aspiration using an echodense tip catheter (Echotip Cook, USA) under ultrasound guidance in 41 patients and the Frydman Catheter (Laboratoire CCD, Paris, France) in 42 patients without ultrasound guidance. Results: Refer to table:

Conclusion: Our data from this prospective study shows that embryo transfers in good prognosis IVF patients, performed under ultrasound guidance with an echodense tip catheter, offers significantly higher implantation and ongoing pregnancy rates in comparison to the use of a semirigid catheter without ultrasound.

P-30 Uterine position at real embryo transfer compared to mock embryo transfer. Melinda B. Henne, Sunny Jun, Amin A. Milki. Stanford Univ, Stanford, CA. Objective: In order to optimize embryo transfer (ET), mock ET is practiced routinely by IVF programs to assess uterine depth and position. The uterine position may vary between a mock ET and the real ET due to the enlarged ovaries in the posterior cul-de-sac, especially when the uterus is retroverted. The purpose of this study is to determine the consistency in uterine position at ET compared to mock ET, depending on whether the uterus is anteverted (A/V) or retroverted (R/V) at mock ET.

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Abstracts

Design: Retrospective data analysis. Materials and Methods: Patients undergoing IVF between January 2001 and September 2002 in the senior author’s practice were evaluated. All had abdominal ultrasound guided mock ET during a natural cycle at initial evaluation. Real ET was performed under abdominal ultrasound guidance with a full bladder. The senior author performed all mock and real ETs. Uterine position was recorded for all patients at the time of mock ET and real ET. Results: During the study period, 636 fresh ETs were performed on 424 patients; 159 ETs were performed on 101 patients with a R/V uterus identified at time of mock ET; 477 ETs were performed on 323 patients with an A/V uterus identified at time of mock ET. At the time of real ET, 88 (55.3%) previously identified R/V uteri converted to an A/V position. Conversely, only 8 (1.7%) previously identified A/V uteri converted to a R/V position. Conclusion: Our data suggest that a R/V uterus at mock ET, will often change position at the time of real ET. A smooth embryo transfer is critical for IVF success. Cramping and disruption of the endometrium from misdirecting the ET catheter are detrimental and can be avoided by accurate knowledge of the uterine position at the time of ET. This information can be more accurately obtained if ultrasound guidance is performed routinely during ET. Additionally, we recommend that patients with a R/V uterus should still have a full bladder for ET, since more than half of them will convert to an A/V position, and will benefit from the bladder effect in straightening the angle of the uterus.

ART: CLINICAL ADJUNCTIVE THERAPIES P-31 Hyperbaric oxygen therapy and in vitro fertilization—A pilot study. Bradley J. Van Voorhis, J. E. Greensmith, Anuja Dokras, Michelle R. Maifeld, Amy E. T. Sparks, Craig H. Syrop. Univ of Iowa Coll of Medicine, Iowa City, IA. Objective: To determine the safety, tolerability and effects of hyperbaric oxygen therapy (HBO) when used during ovarian stimulation for IVF. Angiogenesis and oxygen delivery is critical to the preovulatory ovarian follicle. Impaired angiogenesis in the ovary has been hypothesized to contribute to reduced fertility and increased errors of meiosis in older reproductive age women. Hyperbaric oxygen therapy (HBO) greatly increases tissue oxygen tension and VEGF concentrations, leading to increased angiogenesis in hypoxic tissues. Design: Pilot study of 10 women undergoing IVF treatment. Materials and Methods: Women had to be in a relatively poor prognosis group for IVF; a) age 40 and above or b) age 35-40 with a previously cancelled IVF cycle for poor stimulation. All participants were stimulated with a GnRHa microdose “flare” protocol. HBO dives occured each day (Monday-Friday only) for 2 hours at 2.4 atmospheres of pressure while breathing 100% oxygen. HBO started on the first day of GnRHa administration and continued each weekday until the day of oocyte retrieval. Women were questioned about standard side effects of HBO and IVF. Comparisons of IVF outcomes were made to historical controls (women meeting study entrance criterion in our program in the 6 years prior to the start of this study) and concurrent controls (eligible subjects declining entrance into the HBO arm of the study). At oocyte retreival, one or two pure follicular fluid samples were collected and frozen from all women treated with HBO and from some of the concurrent control women. VEGF concentrations were measured by an immunoassay. Results: The median number of HBO “dives” was 9.5 (range 4-11). HBO was well-tolerated during ovarian stimulation for IVF. One woman dropped out of the study for development of sinus headaches. Otherwise only minor side effects were noted. IVF outcomes are listed in the table. Of 5 women receiving HBO who had an embryo transfer, 2 women are pregnant and both have ongoing twin gestations. Follicular fluid VEGF levels were higher in HBO treated women (n⫽ 10 samples) than in 5 control samples (4649 ⫹ 2055 pg/mL versus 1906 ⫹ 1047 pg/mL, p ⬍ 0.004).

Vol. 80, Suppl. 3, September 2003