Table of Contents
News . . . Sample Issue
Renea Alexander & Boyd Anderson, News Editors
Human Uterine Amylase
Amylase Related to Infertility
Amylase activity in the uterine fluid of infertile women is significantly lower than that in parous women during the different menstrual cycle phases. This low enzyme activity may impair sperm capacitation and adversely affect fertility.
The uterine fluid consists of glandular secretions and endometrial serum transudates. It provides shelter and nourishment to the developing blastocyst. As uterine fluid undergoes cyclic changes during the reproductive cycle, it affects the metabolism and capacitation of sperm during their transport through the female genital tract.
Uterine amylase activity is influenced by endogenous ovarian hormones (Murdock and White, 1969; Skerlavay, Epstein and Sobrero, 1968). These hormones promote the fertilization ability of the sperm. Amylase activity is also negatively correlated with acid phosphatase (Singh and Singh, 1988). Elevated acid phosphatase activity in uterine fluid leads to infertility (Singh, 1991).
V.N. Singh, et al., Bhagalpur University, India, conducted an investigation of 60 healthy women, ages 20-35, with no gynecological problems, (Human Uterine Amylase in Relation to Infertility," Hormonal Metabolism, 1995;27:35-36).
Thirty of these women were considered parous - having had one or more children. The remaining 30 were considered infertile - having had no successful pregnancy even after five or more years of conjugal life. All of these women had normal menstrual cycles and no fallopian tube occlusion. The serum samples from the husbands of the infertile women were collected after 4 days of sexual abstinence; the number, morphology, and the motility of the sperm provided normal values, so were considered fertile.
The uterine fluid samples were collected after 4 or more days of abstinence. The menstrual cycle was divided into 6 phases of four days each, beginning from the 5th day of the cycle. The phases were phase I (early follicular phase - days 5-8), phase II (late follicular phase - days 9-12), phase III (ovulatory phase-days 13-16), phase IV (early luteal phase - days 17-20), phase V (mid-luteal phase - days 21-24), and phase VI (late luteal phase - days 24-28). The amylase activity of the uterine fluid was evaluated using the method of Rice (1959). In the uterine fluid of the parous women, amylase activity showed a significant decrease from Phase I to II, and a significant increase from II to III. Activity declined from III to IV and IV to V. There was a highly significant increase from V to VI. In the infertile women there was no significant change in amylase activity from phase I to II. Activity declined significantly from II to III, then increased significantly from III to IV. From phase IV to V there was a highly significant decrease. Activity highly increased from phase V to VI.
In comparison of the two groups it was noted that the activity level was significantly low at all phases of the menstrual cycle in the infertile women. This could have made their uterine environment nonconductive to sperm capacitation. Endogenous levels of ovarian hormones are reported to affect amylase activity.
The corresponding of this study is V.N. Singh, University Department of Zoology, T.M. Bhagalpur University, Bhagalpur, India 812 007.
Sperm Motility
Anesthesia may Decrease Effect In GIFT
One of the reasons for conflicting pregnancy rates in GIFT may be found in a motility inhibiting substance existing in human peritoneal fluid (hPF). This factor from hPF is a heat-resistant volatile molecule with a very small molecular mass and a possible origin from general anesthesia.
In a study conducted by Dr. W. Miska, University of Giessen, Giessen, Germany, and associates the influence of peritoneal fluid on sperm motility was examined in patients under general anesthesia ("Effect of Anesthesia on Sperm Motility in Gamete Intrafallopian Transfer", Archives of Andrology, Jan./Feb. 1995; 34:43-45). Peritoneal fluids were obtained from the Douglas pouch of women having laparoscopy for infertility work-up. They were in a pre-ovulatory phase and had no peritoneal pathology.
Pre-operatively they received 7.5 mg Midazolam po. General anesthesia was induced with 2 mg vecuronium bromide and 6 mg thiopental /kg body wt. Intubation was facilitated with 1 mg succinylcholine /kg body wt. Then halothane 0.6-1.0% and 66% N(2)O in O(2) was given by controlled ventilation in a nonrebreathing circuit. A slight hyperventilation was maintained.
Spermatozoa were obtained from healthy semen donors with normal semen parameters and purified by the swim-up method in BWW. Separated spermatozoa were incubated with fractions of peritoneal fluid for 30 minutes at 22(degree)C. Sperm motility was measured at 22(degree)C by a computer-aided sperm analysis system (Stromberg-Mida GmbH, Bad Feilnbach, Germany). Motile and immotile spermatozoa were divided in categories according to WHO criteria.
Gamete interactions occur in vivo in the environment of the fallopian tubes. This environment consists of tubal secretions and peritoneal and follicular fluids. Miska writes "Any substance present in hPF with strong influence on the gametes (e.g., inhibiting sperm motility) may be fatal to the success of GIFT. Our results concerning the characterization of a sperm motility-inhibiting factor in hPF indicate a heat-resistant, volatile molecule with a very small molecular mass. This ... factor might be one of the substances that are applied for general anesthesia, such as halothane ... This suggests that the use of local anesthesia(spinal or epidural) for laparoscopic GIFT may confer significant advantages concerning pregnancy rates."
The corresponding author for this study is W. Miska, Department of Dermatology and Andrology, Justus Liebig University of Giessen, Gaffkystrasse 14, 35392 Giessen, Germany.
Luteinizing Hormone
Overexpression of LH Leads to Infertility in Transgenic Mice
Infertility, polycystic ovaries, and ovarian tumors are directly associated with overexpression of luteinizing hormone (LH) in transgenic mice.
A transgenic mouse model has been generated which may prove valuable in studies where excess LH implicates infertility and increased miscarriage rates in women. "The bLH(beta)-CTP transgenic mouse model will be invaluable for these and other studies as it provides a unique opportunity to study the mechanisms whereby elevated LH leads to prolonged luteal life span, cyst formation, tumorigenesis, and perhaps abnormal fertilization and implantation," according to a report in February 1995 issue of Proc. Natl. Acad. Sci., Vol. 92, 1322-1326, by Kimberly A. Risma et al., Case Western University School of Medicine, Cleveland, Ohio.
Fluctuating levels of pituitary gonadotropins: follicle stimulating hormone (FSH) and LH are critical to maintain a pregnancy after fertilization. Correlation of hypersecretion of LH and polycystic ovarian syndrome (PCOS), infertility, and miscarriage in women, suggests that chronically elevated LH impairs fertility, reports Risma et al.
"Unfortunately, there are no studies showing a direct relationship between hypersecreted LH and reproductive abnormalities. Since LH is secreted in regulated pulses and its serum t1/2 is short [20-30 min], it is difficult to devise protocols for chronic administration of exogenous LH that mimic endogenous pulse patterns of LH. To circumvent this limitation, we report a transgenic mouse model in which elevated hormone levels are maintained chronically, without requiring multiple injections, supraphysiologic dosing, or dampening of the hypothalamic-pituitary-gonadal axis," Risma reports.
Two approaches were combined to achieve elevated levels of serum LH. "Increased secretion of hormone from the pituitary was achieved by expression of an LH(beta) subunit transgene, targeted to the pituitary by a previously characterized bovine (alpha)-subunit promoter. The LH(beta) transgene was modified to encode a peptide extension that we proposed would slow the elimination of LH from the serum. This peptide.... is referred to as the C-terminal peptide (CTP). The CTP is thought to be a major determinant of the serum t1/2 of hCG and has been shown to increase the t1/2 of FSH 2- to 3-fold when fused to its (beta) subunit," Risma writes.
Risma et al. reports that serum LH levels were elevated in 13 transgenic (39.7 +/- 8.7 ng/ml; P(less than)0.001) vs. 8 control (2.7 +/- 0.48 ng/ml) females, demonstrating that hypersecretion of LH had been achieved. The transgenic mice ovulated infrequently, maintained a prolonged luteal phase, and developed pathologic ovarian changes such as cyst formation, marked enlargement of ovaries, and granulosa cell tumors. Testosterone and estradiol levels were increased compared to nontransgenic littermates.
"An unusual extragonadal phenotype was also observed: transgenic females developed hydronephropathy and pyelonephritis. The pathology observed demonstrates a direct association between abnormal secretion of LH and infertility and underscores the utility of the transgenic model for studying how excess LH leads to cyst formation, ovarian tumorigenesis, and infertility."
Population Association of America
Age Increases Early Fetal Loss, Not Ability To Conceive
The age-related decline in ability to have children may be due to early fetal loss rather than an inability to conceive, according to a Penn State, University Park, Pennsylvania, anthropologist.
"It appears that age does not affect conception as much as it increases the probability of a high-risk conception that terminates early," Darryl Holman, doctoral candidate in anthropology, told attendees at the annual meeting of the Population Association of America, San Francisco, California, April 8, 1995.
"This increased probability of early fetal loss with increased age suggests that future research in assisted pregnancy might benefit from the careful screening of harvested eggs for genetic defects before in vitro fertilization."
The results of his study indicate that 18 year-old women have a 63.6% chance of fetal loss with each conception, 28 year-old women have an 85% chance of fetal loss with each conception, 38-year-old women, a 95.5% chance of fetal loss with each conception, and 48 year-old women a 98.7% chance of fetal loss with each conception.
"These numbers may seem astonishing, but a 1990 model using in vitro fertilization data came up with an average of about 76%," says Holman.
Working in Bangladesh, Holman carried out a baseline study of 3,200 married women and then a reproductive follow-up survey with 708 women. This nine month-long follow-up survey consisted of twice-weekly visits during which a urine sample was collected and questions on pregnancy, pregnancy sickness and the subject's menstrual cycle were answered.
These 19,000 urine samples from a range of women were processed at the International Centre for Diarrhoeal Disease Research in Bangladesh.
"In the lab we used sensitive chemical assays to detect human chorionic gonadotropin, a hormone secreted by the placenta in pregnant women," says Holman.
The earliest that this hormone can be detected is seven days after conception, when implantation of the fetus takes place.
"The risk of fetal loss follows a pattern," says Holman. "Initially it is very high and drops with time through the pregnancy."
The researchers, who included Dr. James Wood, professor of anthropology, Penn State, and Dr. Kenneth L. Campbell, associate professor of biology, University of Massachusetts, Boston, modeled conceptions as falling into one of two groups, high-risk and low-risk. The high-risk group consists of fetal loss resulting from genetic abnormalities. The low-risk group consists of fetal loss resulting from such events as trauma, infection, placental failure or cervical incompetence. High-risk conceptions lead to fetal loss very early, while the low-risk conceptions usually lead to fetal loss later in pregnancy.
The researchers believe that the increase in fetal loss with age is due to an increase in more high-risk conceptions, and increase in genetic abnormalities in the eggs with age.
Wood's model, based on biological concepts rather than strictly on mathematical principles, modeled these two subgroups of fetal loss to establish the overall risk and effects of age on fetal loss.
"Because this model is biologically based, we were also able to extrapolate back through the initial seven days of pregnancy when existing tests do not identify women as pregnant," says Holman.
Conventional studies have not been able to accurately estimate the number of conceptions because of this seven-day window when women, their doctors and sensitive tests cannot determine pregnancy. These missing seven days appear to be significant in early fetal loss events.
"The percentage of fetal loss that takes place during these seven days is estimated at 24% for an 18 year-old up to 45% for a 45-year-old," says Holman. "A 28 year-old woman has a 37% chance of losing a pregnancy in the first week."
The researchers were able to compare their results to a 1988 study of natural conceptions in U.S. women. They found that Bangladesh women lose only about 5% more pregnancies than do U.S. women.
Understanding the rate of subclinical fetal loss - events that occur without the woman knowing - and the effect that age has on early fetal loss may eventually help to improve the rates of successful in vitro fertilization.
From the anthropological viewpoint, the researchers would like to know how these very early terminated pregnancies affect the birth interval, the time between the birth of one child and the next. This is important because pregnancy hormones take some time to clear the body and early fetal loss may provide a time period during which hormones make ovulation and consequently conception impossible.
The researchers are also testing the urine samples for metabolites of progesterone and estrogen to help determine how long after fetal loss ovulation begins.
Cryopreserved Embryos
Tubal Versus Uterine Transfer of Embryos
"We speculate that tubal transfer of cryopreserved embryos may prove to be the optimal assisted reproductive method in women without tubal occlusion," researchers report.
The first pregnancy achieved after transfer of cryopreserved human embryos was announced in 1983. Since then, cryopreserved embryos have become an important part of assisted reproductive techniques (ART).
Bradley Van Voorhis, M.D., et al. noted a high pregnancy rate in their preliminary experiences with the transfer of cryopreserved embryos to the fallopian tube by laparoscopy (i.e., four pregnancies in six transfers). This laparoscopic tubal transfer is expensive and provides greater medical risk to the patient. "Because of the uncertainty surrounding the efficacy of tubal transfer, we thought it imperative to compare this method of transfer with transcervical transfer of embryos in a prospective randomized trial. This study was designed to test the hypothesis that the pregnancy rate after tubal transfer of cryopreserved embryos is higher than the pregnancy rate after uterine transfer." (Tubal Versus Uterine Transfer of Cryopreserved Embryos: A Prospective Randomized Trial", Fertility and Sterility, March 1995;63(3):578-582).
Nineteen couples were randomly assigned to fallopian transfer and 21 to uterine transfer. Causes of infertility were similar in both groups. There was no significant difference between the two groups in gravidity, parity, age at retrieval, age at transfer, outcome of the retrieval cycle, or endometrial stripe thickness. There was no difference in embryo numbers or quality. Survival of the embryo after thawing was 93% in the fallopian transfer group and 88% in the uterine transfer group. Fallopian transfer was related to a significant increase in the clinical pregnancy rate and the ongoing pregnancy rate. In fallopian transfer the ongoing rate was 58% and only 19% in the uterine. After fallopian transfer the implantation rate was 19.4% and after uterine it was 10.4%.
Van Voorhis summarizes that the results of the study indicate tubal transfer of cryopreserved embryos results in a higher pregnancy rate than uterine transfer in women with patent fallopian tubes and at least 3 cryopreserved embryos. There was no difference in embryo quality as assessed before cryopreservation. In addition, there was no difference in the factors known to influence pregnancy rates. So, none of these factors can account for the increased pregnancy rates observed with the fallopian transfers.
Van Voorhis states, "Tubal transfer of cryopreserved embryos closely mimics a natural conception in terms of early embryo development and implantation. By performing the transfer at the pronuclear stage of development, embryos are in culture for only 26 hours, allowing early embryo development to occur in the fallopian tube. Tubal transfer also allows the embryos to enter the uterine cavity at the appropriate stage of development and precludes any endometrial trauma, which can be associated with uterine transfer."
Therefore the results indicate that "tubal transfer of cryopreserved embryos leads to a significantly higher pregnancy rate than uterine transfer in women with patent fallopian tubes and at least 3 cryopreserved embryos."
The corresponding author of this study is Bradley Van Voorhis, M.D., Bradley Van Voorhis, Department of Obstetrics and Gynecology, University of Iowa Hospitals and Clinics, Iowa City, Iowa 52242-1080.
Improved Pregnancy Rates
Cumulus Removal and Addition of Follicular Fluid
Pregnancy can be achieved without micromanipulation in patients with male factor infertility.
In a recent study Jerome Check et al. evaluated the efficacy of nonmicromanipulation for male factor. Included were two modified in vitro insemination techniques - removal of the cumulus cells with and without the addition of follicular fluid (FF) - in couples with "male factor' problems. (Cumulus Removal and Addition of Follicular Fluid Possibly Improves Pregnancy Rates with In Vitro Fertilization for Male Factor," Archives of Andrology, January 1995;34:47-52).
Check et al. wrote "One of the indications for in vitro fertilization-embryo transfer (IVF-ET) is male factor. However, a problem in assessing the efficacy of IVF for male factor lies in identifying the subfertile male based on merely the semen analysis. Some semen samples with subnormal count, motility, and/or morphology are capable of fertilizing oocytes in vitro, while sperm that appears normal may actually be incapable of resulting in pregnancy. Once the problem has been identified as male factor, the difficulty is in selecting an appropriate treatment." The newer methods of micromanipulation and modified insemination techniques are available when fertilization is not accomplished with conventional methods.
Check notes that it is unclear as to what factors should be included in the definition of severe male factor. "While various semen parameters can indicate a reduced chance of success with fertilization, they cannot accurately predict the outcome following IVF." Their goal was to evaluate the success of patients who had subnormal motility density and subnormal morphology. Their results confirmed that subnormal semen parameters cannot predict poor outcome. Average pregnancy rates presented following IVF-ET.
Check et al. also suggest that treatment for male factor be based on the severity of the problem. High-cost treatments, such as micromanipulation, should be considered only for the most severe. In their program, they "attempted to enhance the cell culture environment to improve the conditions necessary for IVF." This was accomplished by removing the cumulus cells from around the oocytes, pooling more oocytes in a small volume, and adding FF. This removal of cumulus cells may improve the chances of sperm with low or abnormal motility by allowing contact with the zona pellucida. Cumulus removal also allows more pooling of oocytes to increase proximity of sperm and oocytes. "Human FF has been found to contain factors that improve the rate of fertilization of human oocytes in vitro, possibly by enhancing sperm motility, or by providing factors necessary for the completion of the acrosome reaction in vitro".
The study of Check et al. has shown that "pregnancies can be achieved without ... micromanipulation..." Because of the expense of the procedure, they should not be used without discrimination.
The corresponding author of this study is J.H. Check, M.D., 7447 Old York Road, Melrose Park, PA 19027.
Ectopic Pregnancy
Predisposing Risk Factors
Pelvic inflammatory disease and tubal damage have been identified as the two main risk factors for ectopic pregnancy.
The first pregnancy reported after in-vitro fertilization (IVF) was ectopic (Steptoe and Edwards, 1976), and there have been reports ever since of ectopic pregnancies occurring after IVF and embryo transfer.
Because the data on the risk factors associated with ectopic pregnancies after IVF and embryo transfer are conflicting, Samuel Marcus and Peter Brinsden conducted a study to analyze the incidence and risk factors associated with ectopic pregnancy ("Analysis of the Incidence and Risk Factors Associated with Ectopic Pregnancy Following In-Vitro Fertilization and Embryo Transfer," Human Reproduction, January 1995;10(1):199-203).
Data on 135 ectopic pregnancies was taken from records at Bourn Hall Clinic and compared with random samples of 135 singleton deliveries from the same time period.
All of the patients received IVF treatment. Ovarian stimulation was accomplished using either clomiphene citrate and human menopausal gonadotropin with or without follicular-stimulating hormone (FSH) or Luteinizing hormone-releasing hormone agonist buserelin. Ovulation was induced with human chorionic gonadotropin (HCG).
Oocyte retrieval was obtained by vaginal ultrasound-guided method or by laparoscopic or transabdominal recovery. Both fresh and cryopreserved embryo transfer cycles were completed.
Of the 135 ectopic pregnancies there was no evidence of an association between the ectopic pregnancy and a past history of abortion, termination of pregnancy, still birth, neonatal death, or tubal surgery.
There was a positive correlation between a history of pelvic inflammatory disease and ectopic pregnancy.
There was also clear evidence of an association of tubal damage with ectopic pregnancies. A strong association existed between tubal damage and pelvic inflammatory disease. In both normal and ectopic pregnancies the incidence of tubal disease was significantly higher in patients with a history of pelvic inflammatory disease. There was no evidence of a link between the type of ovarian stimulation and ectopic pregnancy. The concentrations of estradiol, LH, and progesterone at the time of ovulation induction did not correlate to ectopic pregnancies. Since the embryo transfer technique did not change during the study period, none of the parameters related to this were analyzed.
There was statistical evidence that the ectopic group received a higher volume of culture medium than the control group. "This result confirms the finding of Knutzen et al. (1989) which showed that after injection of ... radio-opaque fluid through a standard embryo transfer catheter, the material was passed either totally or partially into the Fallopian tubes in 44% of patients, suggesting that the chance of the embryo being carried into the tube immediately after transfer is high. The fact that most ectopic pregnancies occur in patients with damaged tubes supports the hypothesis that the embryo is not transported back to the uterine cavity because of tubal dysfunction".
Samuel Marcus writes, "The overall incidence of ectopic pregnancy after IVF in this series was 135/3000. Although the incidence is comparable with other reports, it is much higher than that following natural conception."
The study also revealed that one in four ectopic pregnancies following IVF and embryo transfer are heterotopic, ovarian or bilateral tubal. Seventy-five percent were single tubal, 15% were heterotopic, 6% ovarian, and 4% were bilateral tubal pregnancies. Marcus states "This should be considered whenever the diagnosis of ectopic pregnancy is made".
In conclusion he adds, "Patients with a history of pelvic inflammatory disease and/or tubal damage are at a higher risk of ectopic pregnancy following IVF and embryo transfer and we recommend injecting the minimum volume of culture medium during embryo transfer."
The corresponding author for this study is Samuel F. Marcus, Bourn Hall Clinic, Bourn, Cambridge CB3 7TR, UK.
Hysterosalpingogram
Determining Diagnostic course After Normal Hysterosalpingogram
Women failing to conceive within one year after a normal hysterosalpingogram demonstrate a high incidence of pelvic pathology with laparoscopy.
To assess the intraluminal environment of the fallopian tube and endometrium the hysterosalpingogram (HSG) is used. However, its ability to evaluate other pelvic pathologies is limited. Laparoscopy (LSC) in conjunction with the HSG will accomplish this. Various studies have indicated the discrepancies between the findings of HSG and LSC in the same patient. Therefore, some researchers recommend LSC as the initial assessment tool.
Most physicians agree that abnormalities discovered with HSC be followed up with LSC and/or hysteroscopy, but there are questions as to what the follow-up should be for patients with a normal HSG.
Bruce Carr M.D. et al., evaluated infertile couples with a normal HSG and assessed their subsequent fertility rates, their laparoscopic findings after a normal HSG, and their historical data ("Infertile Couples with a Normal Hysterosalpingogram," Journal of Reproductive Medicine, January 1995; 40:19-23).
Of the 132 couples with a normal HSG, 29% achieved a pregnancy without having a LSC. There was a fourfold higher pregnancy rate during the first three months post HSG than any other three month period. The pregnancy rate after LSC was 35%.
Carr and associates indicate that their study "supports the hypothesis that women failing to conceive within one year after a normal HSG have a high incidence of pelvic pathology at LSC. If pregnancy did not ensue following a normal HSC, LSC revealed pelvic pathology in 50-60% of cases. Proper timing of LSC is essential ... because of the potential therapeutic effect of HSG." Their study revealed that this effect was achieved by performing HSGs with water-soluble contrast media.
Carr and associates offer some guidelines for timing LSC after a normal HSG. In the women who conceived after HSG but before LSC, the majority of pregnancies occurred in the first three months after the HSG. Also, an increased number of abnormal findings was found on LSC when there was an increase in the time elapsed between the HSG and the LSC.
Some tubal diseases are acquired, so their incidence may increase over time. Their recommendation suggests a waiting period of three months after a normal HSG before performing a LSC. In women who have not conceived by one year after HSG, LSC is warranted because of the high incidence of pelvic pathology.
The corresponding author for this study is Bruce Carr, M.D., Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9032.
Integrins
Assessing Uterine Receptivity
Abnormal endometrial integrins may indicate defective uterine receptivity which may result in unexplained infertility.
The American Fertility Society estimates that 15% to 20% of couples being evaluated for infertility have no identified explanation for their infertility. Unexplained fertility has been attributed to sperm abnormalities, the oocyte, fertilization, ovarian dysfunction, infection, immune problems and peritoneal factors. Because of the lack of acceptable markers for a fertile endometrium there have been few investigations of uterine receptivity defects.
Recently this area has been researched by Bruce Lessey, Ph.D., M.D., and associates ("Integrins as Markers of Uterine Receptivity in Women with Primary Unexplained Infertility," Fertility and Sterility, March 1995;63:535-42). "We described the patterns of expression of integrin cell adhesion molecules in the endometrium of women and noted that one integrin ... abruptly appears in endometrial epithelial cells on post-ovulatory days 5 to 6." The expression of these molecules correlates to the implantation time of the embryo. Previously Lessey and associates demonstrated that histologic delay in endometrium maturation was associated with the loss of a specific integrin. They also demonstrated a relationship between a specific integrin and mild endometriosis. Therefore, integrins may provide a new approach to the assessment of uterine receptivity.
In their study eighty-seven nulliparous women were observed at the Department of Obstetrics and Gynecology at the University of Pennsylvania in Philadelphia or the University of North Carolina at Chapel Hill. All of the women had been previously evaluated for infertility and had been diagnosed with primary infertility of unexplained etiology.
The women's endometrial function was assessed using three cycle-dependent integrins as potential markers of uterine receptivity. (alpha)4 and (beta)3 are subunits that are produced only during maximal uterine receptivity (post-ovulatory days 6-10) so each might be considered potentially useful in detecting endometrial defects. In patients with (alpha)4 missing infertility was not diagnosed. However, (beta)3 proved to be a more reliable marker of the "opening of the window of implantation." (beta)3 is also expressed well into the pregnancy. " The absence of the (beta)3 subunit appears to recognize specifically two distinct pathophysiologic conditions that we have chosen to designate as type I and II defects."
Various studies have suggested that the timing of implantation is from cycle day 20 through day 24. Embryos enter the uterus and implant around post-ovulatory days 4 and 5. If there is a receptive endometrium, the embryo will adhere and implant. If the window of implantation is shifted (as in luteal phase defect or type I defects), or lost (as in Type II defects), the embryo will fail to implant and be lost. Both types of defects reveal distinct mechanisms of defective endometrial receptivity. With type I defects there is a hormonal inadequacy or responsiveness in an otherwise normal endometrium. With type II defects there is a normal menstrual cycle but abnormal endometrial function.
With either defect infertility should be treatable if normal endometrium function can be restored. Therefore, "markers that correctly differentiate such defects could be exceedingly useful in extending our understanding of various infertility states..."
The corresponding author for this study is Bruce Lessey, Ph. D., M.D., Department of Obstetrics and Gynecology, University of North Carolina, MacNider Building, CB 7570, Chapel Hill, North Carolina 27599-7570.
Controlled Ovarian Hyperstimulation
Intrauterine Insemination Versus Timed Intercourse
There is increased incidence of conception in patients treated with controlled ovarian hyperstimulation (COH) and intrauterine insemination (IUI) as opposed to thoses treated with COH and timed intercourse (TI).
A. Gargaropoulos, University of Athens, Areteion Hospital, Athens, Greece, and his associates conducted a crossover study to determine whether there is a benefit from IUI as opposed to TI in ovulated cycles of couples with long standing, unexplained infertility ("Controlled Ovarian Hyperstimulation with or without Intrauterine Insemination for the Treatment of Unexplained Infertility, "International Journal of Gynecology and Obstetrics, 1995;48:55-59).
For the study, forty-six couples were randomly divided into two groups. Each group began one of the two treatment methods: COH/IUI or COH/TI for two consecutive cycles. They then changed to the alternative treatment after a rest cycle, unless pregnancy was achieved. Thus, each patient was her own control. The patients were given hMG for 7 days. If at the end of 7 days there was no increase in serum E(2), the dosage was increased. Human chorionic gonadotropin was injected 24 hours after the last dose of hMg if not more than four leading follicles were seen on ultrasound and the plasma E(2) level was (less than) 1500 pmol/l. The couples were instructed to have intercourse 36-40 hours after the administration of the hCG or insemination was performed approximately 36 hours later.
The couples completed 141 cycles, 67 were with COH/TI and 74 were with COH/IUI. The pregnancy rate after COH/TI was 17.1% and after COH/IUI 45.2%. The difference was significant. The cycle fecundity was 8.9% in those receiving COH/TI and 25.7% in those treated with COH/IUI.
There were no reported signs or symptoms of pelvic infection or significant uterine cramping in the women treated with COH/IUI. Of the 19 pregnancies following IUI, 10 have been delivered and seven are at greater than 20 weeks gestation. Only two spontaneous abortions occurred.
In conclusion Gargaropoulos et al. writes "Although our study indicated favorable results with hMG/IUI treatment over hMG alone ... additional randomized studies will help to clarify the efficacy of hMG/IUI treatment. However, a trial of human menopausal gonadotropin and IUI is justified in couples with prolonged infertility of unknown cause."
The corresponding author of this study is A. Gargaropoulos, 17 Dim. Soutsou Street, Athens 11524, Greece; Tel/FAX: +30 1 6437110.
Negative Post-Coital Tests
In-Vitro Cervical Mucus - Sperm Penetration Tests
In in-vitro cervical mucus-sperm penetration tests (CMSPT), reduced cervical mucus receptivity is a more frequent cause of negative post-coital tests and infertility than is a male factor.
In the in-vivo assessment of sperm-mucus interaction the post-coital test (PCT) is the method generally used. Although it is frequently used, there is little agreement on the interpretation of the results or the optimal mode of treatment for the infertility.
Negative PCT results are associated with various clinical problems and are equated with low pregnancy rates. Further evaluation tools of abnormal PCTs are the in-vitro study of cervical mucus-sperm interaction and cross-hostility tests. By comparing the results of the couple's cervical mucus-sperm penetration test (CMSPT) and the cross-hostility test with the donors' sperm, the cause of the failed sperm-cervical mucus interaction can be better identified.
In their investigation H.S. Jacobs and colleagues evaluated the CMSPT and cross-testing in patients with repeatedly negative PCTs and sought to determine the relationship between the in-vitro test results, different modes of infertility treatment and the pregnancy rate ("In-vitro Cervical Mucus-Sperm Penetration Tests and Outcome of Infertility Treatments in Couples with Repeatedly Negative Post-coital Tests," Human Reproduction January 1995;10(1): 85-90).
"The cervical mucus is the route of access for spermatozoa from the vaginal environment into the upper genital tract and acts as a regulator and filter of sperm transport (Fredricsson and Bjork, 1977). The interaction between the semen and the mucus can be abnormal and lead to infertility. This interaction of male and female secretions can be studied in-vivo using the PCT, but interpretation of the results is difficult and poorly defined. Also, the results have not been proven to correlate with fertility potential. CMSPT has two parameters: distance of penetration and quality of motility. Each of these contribute to the migration of spermatozoa towards the upper female genital tract.
Abnormal PCTs are also evaluated by cross-hostility. This provides a clearer explanation of the factor responsible for the abnormal interaction, be it cervical or the male factor. Positive results from CMSPT correlates with a greater chance of conception.
Jacobs et al. note that their studies "imply that in correctly timed and performed PCTs and CMSPTs, sperm characteristics should be considered the major components that affect the results of penetration tests." In their study only 31% of patients with repeated negative PCTs had a male factor, but 63% of the couples had decreased cervical mucus receptivity. "Stimulation by estrogen on production of cervical mucus results in the secretion of thin, watery cervical mucus, which allows easy penetration by spermatozoa" (Moghissi, 1972; Wold et al., 1978.) "Abnormalities in cervical mucus are believed to be responsible for 5-10% of female infertility" (Balsco, 1977; Roumen et al., 1982).
Jacobs et al. concluded that "Once the limitations of PCT as a qualitative rather than a quantitative study are accepted, the PCT remains an important tool in the evaluation of infertility. If the results ... are repeatedly negative or poor in the absence of proven male factor, the cervical mucus and sperm properties have to be tested not only by CMSPT but should also include a cross-hostility test to determine the etiology of the sperm-cervical mucus interaction."
Jacobs et al. wrote "a decision to progress to assisted reproduction because of a failure of spermatozoa to penetrate cervical mucus in male factor infertility cases should probably be deferred until an attempt has been made to reverse the infertility by treatment with intrauterine insemination. In women with polycystic ovaries and repeatedly negative PCTs, cervical mucus with low quality in terms of sperm receptivity should be suspected. Ovulation induction, together with IUI, should be used ... as the preferred mode of treatment to improve the success rate of infertility treatment."
The corresponding author for this study is Dr. H.S. Jacobs, Cobbold Laboratories, the Middlesex Hospital, Mortimer Street, London W1N 8AA, UK.
Testing/Controversy
The Reliability of Postcoital Test
The postcoital test has been found to have poor to fair reproducibility according to a recent study.
The postcoital test has received widespread use in the evaluation of infertile couples. Many consider it the cornerstone. Because of the controversy over the test's lack of validity for the prediction of pregnancy Mark Hornstein, M.D., and associates have conducted a study to determine the test's reproducibility and to develop a standardized scoring system for the test ("The Reproducibility of the Postcoital Test: A Prospective Study," Obstetrics and Gynecology, March 1995; 85:396-400).
Twenty-eight infertile patients (after 2-3 days of abstinence) were instructed to engage in intercourse 2-12 hours before their examination. They were to refrain from bathing and douching after intercourse. After a standardized collection of post-coital specimens, four reproductive endocrinologists evaluated six postcoital test characteristics and gave their overall impression of the test. The six characteristics were an assessment of the cervical mucus by ferning, cellularity, spinnbarkeit, and consistency, and of sperm by total count and motility.
The results indicated that, except for the determination of sperm number, all other test elements had minimal reproducibility among observers. To further examine the variability Hornstein et al. analyzed the percentage of cases in which all four observers rated the characteristics with the same score. The greatest agreement was in the sperm elements: total number and motility. All four observers gave identical ratings in 39% of the tests. The poorest agreement was in cellularity with perfect agreement in only 11% of the cases.
Each characteristic of the test was also examined to determine their correlation with the observers' score for overall impression. For each observer sperm motility and number were "significantly associated with overall impression." The remaining characteristics were not significantly associated with the overall impression.
According to Hornstein "The postcoital test ... has come under increasing scrutiny in recent years. Concerns that have been raised include a lack of standardization in the test's performance and timing, an absence of agreement in defining the 'normal' range of motile sperm for an adequate test, and its poor sensitivity, specificity, and predictive value for forecasting pregnancy. In addition, test reproducibility has not been defined." The latter was the focus of Hornstein's study. It was demonstrated that "there is poor to fair reproducibility within the individual categories. We question the validity of the postcoital test as a diagnostic tool in the evaluation of infertility."
The corresponding author of this study is Mark Hornstein, M.D., Department of Obstetrics and Gynecology, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115.
Tripterygium
Male Infertility Plant
Six male antifertility diterpene epoxides have been isolated from Tripterygium.
Tripterygium wilfordii Hook. f. (Celastraceae) is a perennial vine growing in southern China. The herb, also called Lei Gong Teng (Thunder God Vine or "three-wing nut"), has been used in Chinese medicine for treatment of fever, edema, and carbuncles for centuries. The powdered roots of the plant were also used as an insecticide.
Over the last 30 years it has been used in the treatment of rheumatoid arthritis, chronic nephritis, chronic hepatitis, thrombocytopenia, ankylosing spondylitis, and skin diseases ("Tripterygium wilfordii, a Chinese herb effective in male fertility regulation" Contraception 1987; 36:335-45).
According to a study by D.Y. Yu in nine rheumatoid arthritis patients treated with Tripterygium for 2 to 56 months, necrospermia or azoospermia occurred.
In experimenting with Wistar rats it was discovered that the seminiferous epithelium was damaged and the serum testosterone levels were decreased when the rats were fed with Tripterygium. In another study rats became infertile after 8 weeks of receiving Tripterygium. Sperm motility decreased sharply and sperm concentration decreased. There was no evidence of toxicity and after 4-5 weeks without the Tripterygium there was full recovery of fertility.
Qian Shao Zhen, Xu Ye, and Zhang Jian Wei observed 26 fertile men being treated with Tripterygium for psoriasis.("Recent Progress in Research on Tripterygium: A Male Antifertility Plant," Contraception, February 1995; 51:117-120). After one month of treatment, the sperm density and motility significantly decreased. After two months of treatment the density and motility decreases further. After one month without the treatment sperm density and motility significantly increased to fertile standards. After another month they were restored to pre-treatment levels. Dr. Zhen et al. concluded that even a small dose of Tripterygium could cause reversible infertility and one of the main sites of action could be the epididymal sperm.
Zhen and associates write that six male antifertility diterpene epoxides have been isolated from Tripterygium. They state "At the ED95 dosage levels, they act mainly on metamorphosing spermatids and testicular and epididymal spermatozoa with exfoliation and inhibition of basic nuclear protein turnover of late spermatids, delayed spermiation and sperm head-tail separation and microtubule, microfilament and membrane damages. A preliminary toxic evaluation indicated that these compounds were immunosuppressive at dose levels 5-12 times their antifertility doses. Immunosuppression is an important weakness for an antifertility agent, but if the immunosuppressive dose of a drug is much higher than its antifertility dose, it could yet be regarded as a safe contraceptive.
The corresponding author of this study is Qian Shao Zhen, M.D., Drum Tower, 19-402 Fifth Lane, Nanjing 210009, P.R. China.
Kallmann's Syndrome
Pregnancy More Than Doubles in Women With Syndrome
More than double the number of pregnancies previously recorded with confirmed Kallmann's syndrome have been obtained in a study published in the March 1995 issue of Fertility and Sterility, by Umit Sungurtekin, M.D. et al., King George V and Royal Prince Alfred Hospitals; Sydney IVF; and University of Sydney, Sydney, New South Wales, Australia.
An association of underdeveloped genitalia and absent olfactory lobes, representing a genetic syndrome, was first described by Kallmann et al. in 1944. Further study by De Morsier and Gauthier lead to a suggested a hypothalamic origin in 1963. Hypogonadotropic hypogonadism with the association of anosmia has been referred to as Kallmann's syndrome, De Morsier's syndrome, olgactogenital syndrome, or olfactogenital dysplasia.
Kallmann's syndrome occurs approximately 1:10,000 in males and 1:50,000 in females. Eight pregnancies in females with Kallmann's syndrome have been previously reported.
Out of six women diagnosed with Kallmann's syndrome from 1974-1994, three wished to undergo induction of ovulation to conceive (ovulation in women with Kallmann's syndrome does not occur without ovulation induction). All six women presented with primary amenorrhea and absence of spontaneous development of secondary sexual characteristics. None of the women complained about the lack of the sense of smell until directly questioned. Anosmia was confirmed with detailed testing.
Human pituitary gonadotropins (hPG) were used for induction until 1985, when hMG superseded the local highly purified extract. Pulsatile GnRH was introduced in 1983.
Ovulation was induced successfully in all three patients, with each patient having multiple conceptions. Of nine pregnancies, there were seven conceptions. One patient had one miscarriage at 6 weeks gestation and a twin pregnancy ending in a pre-term delivery at 29 weeks of gestation. Both infants died due to neonatal sepsis. This patient had previously had a successful single birth and a successful twin pregnancy after the unsuccessful events.
Sungurtekin et al. noted that "the total doses of hPG and hMG used in the pregnancy cycles (mean +/- SEM; 2,850 +/- 375 IU; 40 +/- 5 ampules) and the follicular phase stimulations (mean 15.6 days) were substantially higher than the mean doses used in an age-matched group of women with hypogonadotropic hypogonadism without anosmia (six women; 2,100 +/- 150 IU; 28 +/- 2 ampules; 10.7 days). The follicular phase stimulation with pulsatile GnRH in [one] case (18 and 19 days) were also substantially longer than in our regular program (usually 10 to 14 days)."
"Clearly, more information is required on the range of responses that may occur in women with Kallmann's syndrome. The data currently available are insufficient to make dogmatic statements, and we would like to see data from a standardized GnRH stimulation test in unprimed and GnRH- (and estrogen-) primed women with Kallmann's syndrome compared with their responses to induction of ovulation with pulsatile IV GnRH. In the absence of this definitive predictive information, we currently recommend an initial attempt at induction of ovulation in these women using pulsatile IV GnRH, with the knowledge that some of them will require gonadotropins, usually at a higher dosage than most other hypogonadal patients.
It is self-evident that a patient diagnosed with Kallmann's syndrome should be given estrogen and progestogen therapy for development of secondary sexual characteristics, to mature the uterus, ovaries, and other genital tract tissues, and to protect the bones and cardiovascular system."
Hyperestrogenism
Patients Undergoing IVF with Elevated E(2) Levels at Increased Risk
Because of a report of severe phlebitis and death occurring in two patients after undergoing hyperstimulation, concern exists that patients undergoing IVF techniques resulting in elevated E(2) levels could be at some level of increased risk.
An increased level of coagulation parameters due to pregnancy and oral contraceptive (OC) use has generally been thought to be a result of the estrogen component. Few to minor changes in the hemostatic mechanism have been reported with the newer progestins currently being used in OCs.
Undefined coagulation abnormalities have recently been reported after hMG- and hCG-induced hyperstimulation of the ovary in several women.
A study in the March 1995 issue of Fertility and Sterility, Charles Lox, Ph.D., et al., Texas Tech University Health Sciences Center, Lubbock, Texas, helps to clarify what role high levels of endogenous steroids might play on the potential for thrombosis in women and provides evidence that short-term use of fertility-enhancing drugs and their resultant hyperestrogenism does not induce hypercoagulability.
"We were interested to see if molecular activation of a number of the coagulation factors could be detected after two different IVF regimes that induced high circulation levels of natural endogenous E(2). If evident, this might suggest an increased risk factor for bleeding problems in patients predisposed to thrombosis, for whatever reason," states Lox.
Patients used ranged in age from 26-41 years of age undergoing IVF, with no history of bleeding disorders or coagulation abnormalities, including OC-induced thrombophelebitis or obstetrical bleeding. The first regimen used hMG stimulation only, with the second phase using hMG plus leuprolide acetate (LA). Fifteen patients in part one were stimulated with 150 IU of FSH and 150 IU of LH. Blood samples were collected for E(2) and coagulation factor analysis. The ten patients in part two were down-regulated with LA, 0.2 mL/d, on cycle day 21. After at least a 10-day treatment period or a 3-day withdrawal bleed, LA was decreased to 0.1 mL and hMG was initiated. Only the hepatic synthesized clotting factor activities were evaluated in this part due to costs.
"When looking at both parts of the study, the rise in endogenous E after 10 days of menotropin stimulation was predictable. Levels reached a mean of 1,163 +/- 870 pg/mL (SD), with the maximum level being 2,935 pg/mL. Pooled pretreatment E(2) had a positive correlation with factor VII activity (0.003) and protein C antigen (0.009). After treatment with hMG, the only positive correlation found was with factor V (0.04). There was no significant alteration in either the activity or protein antigen levels before or after 10 days of hMG treatment, nor were the overall measures of hemostatic function altered, the prothrombin (PT) and activated plasma thromboplastin time (APTT)."
"In the second part of the study, LA plus menotropins, changes in E levels were similar to part 1. Levels before hCG ranged from 20 to 4,949 pg/mL while levels after hCG ranged from 26 to 2,287 pg/mL. Progesterone levels in the period before hCG ranged from 0.5 to 4.4 ng/mL and from 0.9 to 204 ng/mL in the period after hCG. The net effect on clotting variables was also minimal. As expected circulating E(2) increased during the period before hCG with a concomitant increase in P during the period after hCG in each patient over the time course of treatment.. All clotting activity increased minimally, although significantly, during the period after hCG except for factor X. Both the PT and APTT minimally decreased in time. All of these changes are still well within the normal ranges for these factors and do not suggest any alteration in the factors that would suggest any type of coagulapathy."
Lox et al. concluded that statistically significant activation of clotting factors occurred during controlled ovarian hyperstimulation (COH). None of these patients experienced any clinically significant coagulation disorder that may be explained by either of the following: [1] the increased levels of clotting activity were still "within normal limits;" [2] none of the patients had any conditions known to predispose to coagulapathies (history of coagulapathies, phlebitis, damaged or compromised endothelial cells, etc.); or [3] a combination of the two.
"Based on the results of this study, our recommendations are that patients predisposed to coagulapathies who also are undergoing COH should be better studied to see if they develop clinically significant clotting activity."
Meiosis-Activating Sterols
Improved Treatment of Infertility, As Well As Hormone-Free Contraception
Researchers at Rigshospitalet University Hospital of Copenhagen, Denmark, in collaboration with Novo Nordisk have discovered a group of compounds that could lead to a treatment for infertility problems, according to Novo Nordisk. Paradoxically, the compounds also seem to be capable of preventing unwanted pregnancy.
The discovery, published in the April 6, 1995, issue of Nature, is the result of collaboration between researchers at Rigshospitalet and Novo Nordisk.
The compounds meiosis-activating sterols (MAS) are present in women's ovaries and in male testes. Under normal circumstances, the compounds will induce the egg cell to complete meiosis, the cell division which results in halving the genetic material. Meiosis is a prerequisite for fertilization of the egg cell. In men, MAS stimulates the formation of sperm cells.
Infertility may be due either to the failure of the egg to divide, or to failing sperm cell formation. And since MAS works on both sexes, the discovery holds promise of offering a unique supplement to hormone therapy offered to infertile couples today.
"I believe that in the next few years MAS could play an important role as a fertility agent for a certain group of people with fertility problems. Especially in the case of in vitro fertilization we believe we will be able to achieve improved results," says Anne Grete Byskov, Doctor of Science, chief of clinic, Laboratory of Reproductive Biology, Rigshopitalet, who heads up the research team.
MAS is different from sex hormones in that it acts on human egg cells and sperm cells only, and apparently has no effect on the rest of the body. Therefore treatment with MAS is believed to have few or no side effects.
With the new knowledge about MAS and its significance for both male and female fertility, it seems likely that it will become possible, in the longer term, to block MAS, thereby preventing unwanted pregnancy. Perhaps we are moving towards a novel, hormone-free contraceptive pill that can be used by both men and women.
"We find this long-term potential for the prevention of pregnancy particularly interesting," says project manager Lars Nordholm, Novo Nordisk, a co-author of the Nature article. "It is our hope that, in collaboration with external partners, we can further develop the project into effective drugs for the treatment of infertility and, later, contraception.
