FERTILITY AND STERILITY~ Vol. 68, No. 3, September 1997

Copyright o 1997 American Society for Repmductive Medicine

Published by Elsevier Science Inc.

Printed on acid-free paper in U. S. A.

Detrimental effect of hydrosalpinx fluid on the development and blastulation of mouse embryos in vitro*

Rahul Sachdev, M.D.? Ekkehard Kemmann, M.D. Michael K. Bohrer, M.D. Iman El-Danasouri, Ph.D.

Department of Obstetrics and Gynecology, University of Medicine and Dentistry of New Jersey (UMDNJ)-Robert Wood Johnson

Medical School, New Brunswick, New Jersey

Objective: To study the effects of human hydrosalpinx fluid on mouse embryo blastula- tion rate.

Design: Comparison of mouse embryo blastulation rate in media containing increasing con- centrations of hydrosalpinx fluid.

Setting: Tertiary care center. Patient(s): Women undergoing laparoscopic evaluation or treatment for infertility noted to

have hydrosalpinx or paratubal cyst. Intervention(s): Exposure of mouse embryos to hydrosalpinx or paratubal cyst fluid collected

during laparoscopy. Main Outcome Measure(s): Blastulation rate of mouse embryos. Result(s): Culture of mouse embryos at 0% (controls), 0.3%, 0.6%, and 0.9% hydrosalpinx

fluid concentrations demonstrated significantly lower blastulation rate at each level compared with the controls.

Conclusion(s): Hydrosalpinx fluid is highly embryotoxic. (Fertil Steril@ 1997;68:531-3. 0 1997 by American Society for Reproductive Medicine.)

Key Words: Hydrosalpinx, in vitro fertilization, embryotoxicity

The development of tubal occlusion with hydrosal- pinx is a major cause of infertility. This condition usually is associated with a previous pelvic infection ( 1 - 3), and, frequently, optimal surgical correction is not possible (4, 5). To achieve a successful preg- nancy, IVF-ET has become the major option for these women. Recently, several reports have demon- strated significantly lower pregnancy rates (PRs) and higher risk of pregnancy loss with IVF-ET in the presence of a unilateral or bilateral hydrosalpinx (6-8).

Fluid from the hydrosalpinx may leak into and

Received November 26, 1996; revised and accepted May 14, 1997.

* Presented at the 52nd Annual Meeting of the American Soci- ety for Reproductive Medicine, Boston, Massachusetts, November 2 to 6, 1996.

i Reprint requests: RahuI Sachdev, M.D., UMDNJ-RWJ, De- partment of Obstetrics and Gynecology, 303 George Street, Suite 250, New Brunswick, New Jersey 08901 (FAX 908-235-7318).

0015-0282/97/$17.00 PI1 SOO15-0282(97)00238-O

accumulate in the uterine cavity (9). It is unclear whether this creates a hostile local environment in the uterus for embryo implantation (10,111 or exerts a direct embryotoxic effect. Surgical correction or re- moval of the hydrosalpinx restores IVF-ET PRs com- parable to those without tubal disease (8).

The aim of this study was to investigate the effect of human hydrosalpinx fluid on the development and blastulation of mouse embryos.

MATERIALS AND METHODS

This study was approved by the Robert Wood Johnson Medical School Institutional Review Board. Fluid was collected from five women with unilateral or bilateral hydrosalpinx at the time of laparoscopic surgery by needle aspiration at Robert Wood John- son University Hospital. One additional patient had fluid collected from a paratubal cyst. Four of the five women in the hydrosalpinx fluid group had a history

531

Table 1 Effect of Hydrosalpinx Fluid Concentration on Blastocyst Formation

Percent hydrosalpinx fluid

0 0.3% 0.6% 0.9% ~ ~ -

Patient ABABABAB

1 17 12 20 10 20 10 2 22 19 22 11 23 0 3 17 15 20 13 18 0 4 20 20 20 10 20 0 5 22 21 23 12 22 0

Total zygotes 98 105 103 Total blastocysts 87 56 10 Percent

blastulation 88.8 53.3* 9.7t

21 0 20 0 18 0 20 0 20 0 99

0

O$

Note: A columns are the number of embryos cultured at the HF concentration. B columns are the number reaching blastocyst stage.

* P < 0.05 compared with 0 (controls), using the Fisher’s exact test.

t P < 0.05 compared with 0 (controls), using the Fisher’s exact test.

j: P < 0.05 compared with 0 (controls), using the Fisher’s exact test.

of chlamydial exposure and long-term infertility. The patient with the paratubal cyst did not have a history of chlamydial exposure. Upon collection, hydrosalpinx fluid was centrifuged to remove any cellular debris, filtered through a 0.22 pm filter and frozen at -80°C until use. At time of the experi- ments, hydrosalpinx fluid was thawed and osmolar- ity and pH were measured.

Embryos were obtained by superovulating C57BL x 6J female hybrid mice at 4 to 6 weeks of age. Mice were injected intraperitoneally with 5 IU pregnant mare serum gonadotropin (PMSG; Sigma Chemical Co., St. Louis, MO). Ovulation was induced 48 hours later by administering 5 IU hCG (Sigma). Animals then were placed overnight with 15week-old males, and mating was confirmed by the presence of a vagi- nal plug the next morning. Females were killed, and cumulus-enclosed zygotes were recovered from the oviducts and immediately transferred to human tubal fluid medium (HTF; Irvine Scientific, Santa Ana, CA) containing 5 mg/mL bovine serum al- bumin.

Preliminary experiments demonstrated that none of the embryos survived to blastocyst stage when cultured at 2 1% hydrosalpinx fluid concentration. Hence, in this experiment, zygotes were washed and transferred into 30 PL microdrop culture media con- taining only HTF (controls) or 0.3%, 0.6%, or 0.9% hydrosalpinx fluid (study group). Similarly, experi- ments were performed with fluid from the paratubal cyst at the same concentrations. Embryos were ex- amined daily to record the number reaching ex-

panded blastocyst stage. Comparisons were made between the control and the study groups using the Fisher’s exact test to detect any difference in the blastulation rate.

RESULTS

The mean pH and osmolarity of the thawed hy- drosalpinx fluid was 7.6 and 293 mOsm, respec- tively. Table 1 demonstrates a decline in blastocyst formation with increasing hydrosalpinx fluid concen- tration for all five patients. Similarly, the cumula- tive data demonstrate a significant decline in em- bryo blastulation rate with increasing hydrosalpinx fluid concentrations using the Fisher’s exact test.

The pH and osmolarity of the paratubal cyst fluid were in the physiologic range. Incubation of the em- bryos with this fluid in increasing concentrations did not demonstrate a significant decline in the blastula- tion rate and, thus, no detrimental effect (Table 2).

DISCUSSION

This study demonstrates the highly embryotoxic nature of hydrosalpinx fluid. At low concentrations between 0.3% and 0.9%, there is a significant decline in the mouse embryo blastulation rate. None of the embryos survived to the blastocyst stage at 20.9% hydrosalpinx fluid concentration. In contrast, fluid from the paratubal cyst demonstrated no such toxic- ity, suggesting that tubal processes unique to the hydrosalpinx are involved in the pathogenesis of em- bryotoxicity. Previous reports of reduced IVF-ET PRs in the presence of hydrosalpinx (6-8) and those demonstrating a restoration of higher PRs after cor- rection of hydrosalpinx (8) are consistent with our

Table 2 Effect of Paratubal Cyst Fluid on Blastocyst Formation

Percent paratubal cyst fluid

0 0.3% 0.6% 0.9% - - - -

A B A B A B A B

25 23 20 17 20 16 23 18 Percent

blastulation 92 85* 8Ot 78%

Note: A columns are embryos cultured at the paratubal cyst fluid concentration. B columns are the number reaching blasto- cyst stage.

* No significant decline in blastulation rate compared with 0 (controls) using the Fisher’s exact test.

t No significant decline in blastulation rate compared with 0 (controls) using the Fisher’s exact test.

$ No significant decline in blastulation rate compared with 0 (controls) using the Fisher’s exact test.

532 Sachdev et al. Hydrosalpinx fluid is embryotoxic Fertility and Sterility@

finding of the embryotoxic nature of hydrosalpinx fluid. Indeed, the removal or correction of the hy- drosalpinx eliminates the source of the toxicity allowing the embryos to implant. Additionally, the hormonal therapy during IVF-ET stimulation proto- cols may stimulate tubal secretions, leading to an increased hydrosalpinx size (12). This may further create an unfavorable environment for the embryo in utero.

Contact between hydrosalpinx fluid and embryos in utero is possible, and reported cases exist (9) dem- onstrating accumulation of fluid in uterine cavity in the presence of a hydrosalpinx. Additionally, uterine ostium may not need to be fully occluded for a hy- drosalpinx to develop (13). The highly toxic nature of hydrosalpinx fluid may require only a small vol- ume of fluid to be present in utero to exert its detri- mental effects.

Report of higher pregnancy loss (10) in the pres- ence of hydrosalpinx seems to suggest an additional effect of hydrosalpinx fluid at the level of the endo- metrium. Quite possibly, endometrial function or maturation process may be harmed, leading to preg- nancy loss. Hence, overall, hydrosalpinx creates a highly unfavorable uterine environment for concep- tion to occur.

There is a paucity of data regarding the composi- tion of hydrosalpinx fluid. It has been reported that the electrolyte concentrations are similar to serum, but protein and bicarbonate levels are lower (14). Because hydrosalpinx typically occurs after an infec- tious process, this fluid may contain cytokines, leu- kotrienes, prostaglandins, or other components that may be detrimental to the embryo (15). HF pH should not be an issue, as our study demonstrated it to be in the physiologic range. Further studies to determine what the embryotoxic component(s) may need to be performed.

In view of the previous studies and our confirma- tion of the highly embryotoxic nature of hydrosal- pinx fluid, one may want to consider repairing the fallopian tube(s) surgically or performing a salpin- gectomy before proceeding with IVF-ET to maximize the PR. Current data (16) suggest that performing a salpingectomy does not compromise ovarian stimu-

lation during IVF and should not be an impediment for treatment to maximize the PR with IVF-ET.

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