Utility of in vitro fertilization at diagnostic laparoscopy*

FERTILITY AND STERILITY Copyright" 1994 The American Fertility Society Printed on acid-free paper in U. S. A. Utility of in vitro fertilization at diagnostic laparoscopy* Paul R. Gindoff, M.D.t Jerry L. Hall, Ph.D. Robert J. Stillman, M.D. Department of Obstetrics and Gynecology, The George Washington University Medical Center, Washington, D.C. Objective: To compare stimulation and outcome variables for IVF in stimulated cycles when ova are retrieved during diagnostic infertility laparoscopy versus transvaginal ultrasound (US) directed retrieval and to investigate the presence of unexpected failed fertilization in the diagnostic laparoscopy group, which allows an opportunity to diagnosis an etiology of infertility based on gamete interaction. Design: Consecutive patients who needed infertility diagnostic laparoscopy and agreed to combination with IVF were compared with concurrent patients undergoing transvaginal US IVF. Male factor screening parameters (semen analysis, sperm penetrating assay) and resultant fertilization were analyzed for these patients. Setting: The George Washington University Hospital, a tertiary referral center offering assisted reproductive technologies. Patients for diagnostic laparoscopy combined with IVF were enrolled in the Program of Oocyte Retrieval at Diagnostic Laparoscopy (). Participants: One hundred twenty-four women enrolled for diagnostic laparoscopy combined with IVF; 237 women were concurrently enrolled for transvaginal US IVF. Results: Response variables (number of follicles, days of monitoring, ampules of hmg, maximum E2) between the two groups were similar. Outcome variables (ova retrieved, ova fertilized, ova cleaved, clinical pregnancy rate per embryo transferred) were similar despite a significantly higher number of embryos transferred for the transvaginal US group. The clinical pregnancy rate per cycle was similar, 26% versus 28% for the women in the transvaginal US versus those women in the group, as was the clinical pregnancy rate per ET, 31% versus 34%, respectively. The number of fertilized ova for each group was not significantly different: 5.5 ± 0.3 for the transvaginal group versus 4.8 ± 0.4 for the group. Patients in the group with a known male factor (group B) had a lower fertilization rate than those with no male factor (group A). Within the group A with no detectable male factor prospectively, 17.2% had unexpectedly poor fertilization (group AI)' whereas the rest of the group A patients had higher fertilization rates as was expected (group A2). The clinical pregnancy rate per ET for group Al was 0% compared with 43.4% for the group A2 patients. Conclusions: In vitro fertilization can be successfully performed during diagnostic laparoscopy yielding comparable results to transvaginal ultrasound IVF while gaining diagnostic information concerning sperm-ova interaction (i.e., fertilization). Fertil SterilI994;62:237-41 Key Words: IVF at diagnostic laparoscopy, ART, diagnosis Received September 22, 1993; revised and accepted April 13, 1994. * Presented in part at the Panel on "Life cycles of Reproductive Health" during the 48th Annual Meeting of The American Fertility Society, New Orleans, Louisiana, November 2 to 5, 1992. Laparoscopy is a fundamental component of the evaluation of the infertile woman. Assisted reproductive technology (ART) can be performed successfully during diagnostic infertility laparoscopy (1-3). The combination of diagnostic laparoscopy t Reprint requests: Paul R. Gindoff, M.D., Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, The George Washington University Medical Center, 2150 Pennsylvania Avenue, NW, Washington, D.C. 20037 (FAX: 617-350-8315). Gindoft' et ai. IVF at diagnostic laparoscopy 237

with laparoscopic oocyte retrieval for ART offers the following advantages: diagnosis of the cause of infertility may be established; therapy may be given to improve conception rate of subsequent cycles, that is, pelviscopic therapy (4); and conception may be achieved in the actual cycle by IVF -ET or GIFT. Furthermore, operative endoscopic manipulation and therapy does not adversely influence pregnancy outcome of ART when the two are done in combination (1). Another advantage of combined diagnostic laparoscopy and IVF -ET is the opportunity to examine the oocyte and sperm interaction in vitro. Unexpected failed fertilization of oocytes may suggest a problem with gamete interaction (i.e., fertilization factor) despite normal andrology screening. In the present study, we compared the results in two concurrent groups of patients subjected to identical stimulation protocols undergoing subsequent IVF ET. One group of patients underwent diagnostic infertility laparoscopy with oocyte recovery via laparoscopy; the other group underwent oocyte recovery by transvaginal ultrasound (US)-guided needle aspiration. Demographic variables, response variables, and outcome parameters were compared between the two groups. Patient Selection MATERIALS AND METHODS Infertile women were eligible for inclusion in the Program of Oocyte Retrieval with Diagnostic Laparoscopy () at The George Washington University Medical Center if indications existed for diagnostic laparoscopy. They were empirically offered the option of ART during the cycle of their laparoscopy. We excluded those who, based on preoperative hysterosalpingogram, required intraoperative chromotubation or hysteroscopy. After providing appropriate informed written consent, 124 patient enrolled in the program were compared with 237 women who concurrently initiated a cycle protocol for transvaginal US-guided needle aspiration of oocytes for IVF. In vitro fertilization was recommended to patients who were already evaluated completely (including laparoscopy) and had a diagnosis (i.e., tubal factor, male factor) that required IVF as therapy. Protocol General anesthesia was used for laparoscopy, which included nitrous oxide and enflurane in con- junction with muscle relaxants initiated after sterile abdominal preparation and draping. Ultrasound procedures were done under local lidocaine analgesia and intravenous narcotic sedation and oral diazepam. The ovulation induction protocol, identical for both groups, was preceded by luteal phase-initiated leuprolide acetate (LA) (Lupron: TAP Pharmaceuticals, Chicago, IL) suppression. Leuprolide acetate was begun in the luteal phase (day 23) and administered for 10 days (5). Evaluation of the ovaries by transvaginal ultrasound (UItramark IV; ATL, Bothall, WA) and serum E2 was done to assess if ovarian suppression was achieved. After documentation of LA suppression, stimulation was initiated with two ampules of hmg twice daily (Pergonal; Serono, Inc., Randolph, MA) for 5 days (5). The stimulation was then individualized by monitoring with daily vaginal sonography and serum E2. In all subjects, hcg (10,000 IU) was administered 1M 35.5 hours before oocyte recovery when E2 levels reached or exceeded 500 pg/ml (conversion factor to SI unit, 3.671) with two or more follicles attaining a mean diameter of 16 mm or larger. During the diagnostic laparoscopy, both tubal and ovarian adhesions and implants of endometriosis were treated by pelviscopic techniques, including lysis with endoscopic scissors or electrocautery, respectively. Standard aspiration techniques at laparoscopy or with transvaginal US and laboratory techniques of oocyte handling have been published previously (1, 5). After oocyte retrieval, semen samples were processed by swim-up technique (6) coinciding with timing for in vitro insemination of recovered oocytes. Luteal support was maintained by three hcg injections of 3,000 IU every 3 days over the luteal phase. Pregnancy results reported are clinical, documented by US detection of fetal heart activity in addition to rising titers of j1-hcg. Precycle Screening Andrology Evaluation The male partner had been evaluated with at least one semen analysis and semen wash (6) using the swim-up technique. If no recent prior pregnancy had been fathered, a sperm penetration assay (SPA) was also performed (7). A normal SPA was >19%. A normal semen analysis was defined as a sperm concentration of >20 X 10 6 /ml and a motility> 40% (Society of Assisted Reproductive Technology guidelines for male factor) with normal morphology > 60% (World Health Organization guidelines) (8). 238 Gindoff et al. IVF at diagnostic laparoscopy Fertility and Sterility

Statistical Analysis Comparative group data was analyzed by Student's t-test. Values are presented as means ± SE. Pregnancy rates were compared by x 2 analysis. In all cases, significance was taken at the P < 0.05 level. Assays Serum E2 levels were measured with RIA kits on a stat basis (Diagnostic Assay Services, Gaithersburg, MD). RESULTS The demographic variables and distribution of patients in the two groups, transvaginal-us and, are presented in Tables 1 and 2. Cancellations for each group were due to poor response to ovulation induction. Table 2 further highlights the response parameters for the two groups. Mean age for transvaginal-us versus (36.0 ± 0.3 vs. 35.4 ± 0.4, P > 0.05, respectively) were similar. Both groups reached similar peak E2 concentration levels (1,109.7 ± 46.0 vs. 1,175.4 ± 74.3 pg/ml, P > 0.05, transvaginal-us vs., respectively) and had a similar number of follicles (> 14 mm) develop on US (4.6 ± 0.2 vs. 4.4 ± 0.3, P> 0.05, transvaginal-us vs., respectively). The days of monitoring (3.2 ± 0.1 vs. 3.3 ± 0.1, P > 0.05, transvaginal-us vs., respectively) and required ampules of hmg needed (35.7 ± 0.9 vs. 34.0 ± 1.2, respectively, P > 0.05) were similar. Table 3 details the outcome variables to ovulation induction with the LA suppression protocol in these patients. There were similar results in the number of ova Table 2 Demographic and Response Variables Patients stimulated 237 124 Cancellations* 28 (12) 20 (16) Age (y)t 36.0 ± 0.3 35.4 ± 0.4 E2 maxt (pg/ml):j: 1109.7 ± 46.0 1175.4 ± 74.3 No. follicles (>14 mm mean diameter)t 4.6± 0.2 4.4 ± 0.3 Days of monitoringt 3.2 ± 0.1 3.3 ± 0.1 Ampules of hmgt 35.7 ± 0.9 34.0 ± 1.2 Days to suppress with GnRH-at 14.8 ± 0.5 15.1 ± 0.9 * P > 0.05, NS by x2 analysis. Values in parentheses are percents. t P > 0.05, NS by t-test. Values are means ± SE. :j: Conversion factor to SI unit, 3.67. retrieved (7.0 ± 0.3 vs. 6.6 ± 0.5, P > 0.05), ova fertilized (5.5 ± 0.3 vs. 4.8 ± 0.4, P > 0.05) and ova cleaved (4.6 ± 0.2 vs. 4.0 ± 0.4, P > 0.05) for transvaginal-us versus, respectively. Despite the significantly higher number of embryos available for transfer in the transvaginal US group compared with the diagnostic laparoscopy group (3.2 ± 0.1 vs. 2.8 ± 0.2, P > 0.05, transvaginal-us vs., respectively), the clinical pregnancy rates per cycle, retrieval, and ET were not significantly different between the two groups (Table 3). To analyze the value of prescreening semen parameters on IVF success in patients, the 104 patients who underwent laparoscopic oocyte retrieval were divided into groups based on their andrology testing. Group A consisted of the 67 patients with normal semen analysis and/or SPA. Group B consisted of the 37 patients with an abnormal semen analysis and/or SPA. The average was Table 1 Distribution of Patients by Primary Diagnosis Tubal! adhesions Endometriosis Unexplained infertility Male factor 52 10 19 19 * Final diagnosis made at laparoscopy. t Negative laparoscopy along with negative work-up yielded unexplained infertility as diagnosis. :j: Of the 104 patients that had laparoscopy, 9 had negative laparoscopy but abnormal precycle andrology screening tests for male factor as primary diagnosis (9%). An additional 28 patients had abnormal precycle screening andrology tests for a secondary diagnosis of male factor. % 38* 36* 17t 9:j: Table 3 Outcome Variables Ova retrieved per patient* 8.8 ± 0.4 Mature ova per patient* 7.0 ± 0.3 Ova fertilized per patient* 5.5 ± 0.3 Ova cleaved per patient* 4.6 ± 0.2 No. embryos transferredt 3.2 ± 0.1 per cycle:j: (%) 26 per retrieval:j: (%) 27 per ET:j: (%) 31 * P > 0.05, NS by t-test. Values are means ± SE. t P < 0.05, by t-test. Values are means ± SE. :j: P > 0.05, NS by x2 analysis. 8.5 ± 0.5 6.6 ± 0.5 4.8 ± 0.4 4.0 ± 0.4 2.8 ± 0.2 28 32 34 Gindoff et al. IVF at diagnostic laparoscopy 239

Table 4 Influence of Andrology Screening on Outcome Group A: Group B: Normal Abnormal Screening Screening S/A and/or SPA S/A and/or SPA No. of patients 67 37 No. of ova retrieved* 7.6 ± 0.6 7.4 ± 0.8 Fertilization ratet (%) 63.1 43.0 No. of clinical pregnancies 23 10 Clinical pregnancies per retrieval* (%) 34.0 27 Clinical pregnancies per ET* (%) 35.9 29.4 * P > 0.05, NS, by t-test. Values are means ± SE. t P < 0.0001 by t-test. * P > 0.05, NS, by x 2 analysis. 36 ± 3.6 years versus 34.5 ± 4.1, A versus B, respectively (P > 0.05), and the average duration of infertility was 3.7 ± 2.7 years versus 3.8 ± 2.4 years, A versus B, respectively (P> 0.05). There were three failed fertilizations with no ET for each group. Table 4 details the outcome of IVF for these two groups of patients. Fertilization rates occurred in 63.1 % of all oocytes recovered for group A versus 43.5% of all oocytes recovered for group B (P < 0.0001). rate per retrieval and clinical pregnancy rate per ET were 34 % versus 27% and 35.9% versus 29.4% (A versus B, respectively, not significant, P > 0.05, respectively. However, among the 67 patients in group A with normal precycle screening andrology tests, 11 of these patients had an unexpectedly low fertilization rate of <33% (group AI). The remaining 56 patients in group A had a higher fertilization rate (group A2). All pregnancies occurred in this subgroup (group A2), whereas all the cycles ending up with no ET due to no fertilization of ova occurred in the subgroup AI. The clinical pregnancy rate per ET in those patients with normal andrology screening yet poor fertilization (group AI) was 0% compared with 43.4% for the group A2 patients (P < 0.01). DISCUSSION This study confirms and expands our earlier report regarding the feasibility and efficacy of performing ART successfully during diagnostic infertility laparoscopy (1). The efficacy of ART, when combined with laparoscopy, is not decreased compared with the transvaginal US-needle guided oocyte aspiration approach. The ability to perform therapeutic operative procedures during diagnostic laparoscopy is retained. This is associated with an increased cost as compared with pelviscopy alone, but can be associated with an increased therapeutic advantage and diagnostic edge. Thus, three goals may be realized: laparoscopic diagnostic information is obtained, laparoscopic surgery as a therapeutic modality is retained, and attempted conception utilizing ART during the cycle of surgery may be achieved. In addition, potential diagnostic information concerning fertilization difficulties due to previously undiagnosed male factor or ova factor is evidenced by evaluating in vitro sperm-oocyte intera,ction (i.e., fertilization). Male patients found to have an abnormal semen analysis and/or SPA on precycle screening had lower fertilization rates in vitro. However, their overall pregnancy rates were not different from males with normal precycle andrology studies. This may have been due to precycle planning for different handling of the sperm and ova in the laboratory for a known male factor. Of interest was the unexpectedly low fertilization rate in a subgroup of patients with normal precycle andrology screening who underwent attempted IVF without special treatments for male factor. For this group, diagnostic information was obtained by documenting a problem with fertilization (sperm-ova interaction). The recognition of this problem in ova fertilization requires laboratory observation of gametes in culture that may not Qe possible if GIFT is performed, leaving at most, only supernumerary ova that may be suboptimal for this purpose. Although this study does not document that this poor fertilization was due to a male factor exclusively, the fact that the patient's age and the number of mature oocytes were similar in the groups A and B (with and without normal andrology testing, respectively) suggests that the ova quality may have been similar in both groups. Therefore, when it is decided to use ART at the time of diagnostic laparoscopy, we recommend IVF rather than GIFT for both diagnostic and possible therapeutic advantage. This, of course, presumes the appropriate laboratory capabilities for successful fertilization and pre-embryo culture in vitro. Compared with laparoscopic procedures, transvaginal oocyte retrieval under US guidance encompasses less risk, is more economical in terms of time and costs, and may be performed without general anesthesia. As the trend toward increased utilization of transvaginal US guidance for oocyte recovery has increased, simultaneously, there has been some increase in the overall efficacy of IVF as 240 Gindoff et al. IVF at diagnostic laparoscopy Fertility and Sterility

reported in the literature (9). In general, it is considered the method of choice for oocyte recovery for IVF -ET. However, should the patient require a laparoscopy for evaluation or treatment of infertility, a reasonable option, despite the additional cost to the patient, may be to take advantage of the combined procedure of ova harvesting at the same time for use in assisted reproduction. Acknowledgment. Special thanks to Ms. Tracey Craddock for her expertise in manuscript preparation. REFERENCES 1. Gindoff PR, Hall JL, Nelson LM, Stillman RJ. Efficacy of assisted reproductive technology during diagnostic and operative infertility laparoscopy. Obstet Gynecol 1990;75:299-301. 2. Shoham Z, Barash A, Yemeni M, Borenstein R. Combined diagnostic laparoscopy and follicular aspiration for human in vitro fertilization. Acta Obstet Gynecol Scand 1990; 69:23-6. 3. Barad D, Bartfai G, Barg P, Cohen BL, Feinman M. Ga- mete intrafallopian tube transfer (GIFT): making laparoscopy more than "diagnostic." Fertil Steril 1988;50:928-30. 4. Damewood MD, Rock JA. Treatment of independent pregnancy with operative laparoscopy for endometriosis in an in vitro fertilization program. Fertil Steril 1988;50:463-5. 5. Gindoff PR, Hall JL, Stillman RJ. Ovarian suppression with leuprolide acetate: comparison of luteal, follicular and administration in controlled ovarian hyperstimulation for oocyte retrieval. J In Vitro Fert Embryo Transfer 1990; 7:94-7. 6. Asch RH, Balmaceda, Ellsworth LR, Wong PC. Preliminary experience with gamete intrafallopian transfer (GIFT). Fertil Steril1986;45:366-71. 7. Hall JL. Relationship between semen quality and human sperm penetration of zona-free hamster ova. Fertil Steril 1981;35:457-63. 8. World Health Organization. Laboratory manual for the examination of human semell and semen-cervical mucus interaction. In: Belsey MA, Eliasson R, Gallegos AJ, Moghissi KS, Paulson CA, Prasad MRN, editors. Singapore: Press Concern, 1980;1-25. 9. Medical Research International. In vitro fertilization-embryo transfer (lvf-et) in the United States: 1990 results from the IVF-ET Registry. Fertil Steril1992;57:15-24. Gindoff et al. IVF at diagnostic laparoscopy 241