Evaluation and Preparation of the Infertile Couple for In Vitro Fertilization

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David R. Meldrum

Reproductive Partners Medical Group, Redondo Beach, California, U.S.A.

Thorough evaluation of the infertile couple before in vitro fertilization (IVF) is critical in achieving the best outcomes and avoiding complications. Most IVF centers organize the evaluation by using a checklist that the nurse coordinator and physician assure is complete before proceeding with the cycle.


A level exceeding 25mIU/ml (about 12mIU/ml using current assays) has been correlated with a very low chance of pregnancy (1). More recent studies have shown that mild elevations in women below 40 yr of age predict a more modest reduction in the pregnancy rate, whereas an elevated level carries much more meaning in older women. Sometimes, particularly in older women, follicular maturation is very rapid and the follicle-stimulating hormone (FSH) can already be decreasing by day 3. Therefore, the level of estradiol (E2) should also be measured. The impact of an increased day 3 E2 level (over 70-80 pg/ml) in the presence of a normal FSH concentration is unclear, but using gonadotropin releasing hormone (GnRH) agonist and assisted hatching, an elevated E2 level correlates with increased cycle cancellation but not with a reduced pregnancy rate (2). An elevated day 3 E2 has less importance in young women. We also use the FSH assay to predict the optimal level of stimulation, because the ovarian response has been shown to vary inversely with the FSH level (1). For women with an FSH level over 10mIU/ml, we generally choose a low responder protocol.

FSH assays vary considerably in their normal ranges. Ideally, if switching from the Leeco Diagnostics, Southfield, Michigan Company, (now Binax, Inc., Scarborough, Maine) assay on which older research was based (1), a series of samples should be run in parallel using both methods, so that the new assay levels can be interpreted appropriately. In our case, when switching to the Immulite (DPC) system, a level of 12mIU/ml corresponded to 25mIU/ml in the BINAX system. In the absence of such direct comparison, one can use the College of American Pathologists survey booklet, which gives mean levels for all labs using each kit and standard sera.

FSH levels also vary from cycle to cycle. A consistently elevated FSH predicts a poorer prognosis than a single elevated level with others being normal. The quality of ovarian stimulation is not improved when IVF is done in a cycle with a more normal FSH level (3). There is an agreement that women with a single elevated FSH level have a high cancellation rate, but studies conflict regarding the extent of reduction of pregnancy outcome (4,5). FSH levels are similar on days 2, 3, and 4. Women with premenstrual spotting should be advised to count the first day of full flow as day 1.


It is the antral follicles that respond to stimulation. With a high-quality transvaginal ultrasound scan, these can be accurately counted. Follicle count decreases with age in normal women (6). In women with 5-10 follicles per side, one expects a normal response to stimulation. With more than 10 per side [polycystic ovary (PCO)-like], a lower level of stimulation should be chosen than otherwise would be used based on weight and FSH level. A low follicle count (fewer than 5 or 6 in total) predicts a lower prognosis (7,8) and should prompt a higher level of stimulation. Total follicle count correlates positively with the number of oocytes retrieved and negatively with day 3 FSH and ampoules of gonadotrophins, with fewer than 10 total follicles predicting an increased chance of cancellation (9). By multivariate analysis, antral follicle count was found to be the best single predictor of ovarian response and therefore prognosis, with FSH having a small additive effect (10). As the outcome of IVF is very low in women above 40 years of age who develop fewer than three follicles with stimulation (11), a low resting follicle count can be used together with other data (age, day 3 FSH, duration of infertility) to suggest egg donation as a better option.


The clomiphene citrate challenge test (CCCT) has been used to identify patients with a low prognosis and low ovarian reserve who have a normal day 3 FSH level (12). CC is taken at 100 mg/day from days 5 to 9. The day 10 FSH level should be less than 10-12 mlU/ml. In normal women, although FSH is stimulated by CC, the rising E2 brings it back into the normal range. In women with low ovarian reserve, the pituitary responds with a more prominent FSH rise which is not suppressed as promptly by the rising E2 level. Clearly there is a group of women with a normal day 3 FSH with an abnormal CCCT who have a reduced prognosis, but in the usual instance the couple will choose to go ahead regardless, and other information such as the antral follicle count will be sufficient to plan the ovarian stimulation. A recent study looking at various markers of ovarian reserve failed to find any clear additional predictive value for poor ovarian response in addition to FSH and antral follicle count (13).


Women with PCO produce more follicles with stimulation. More oocytes are retrieved, having a lower fertilization rate. The pregnancy rate is as good as other women having IVF. Provided a GnRH agonist is used, the miscarriage rate is normal (14).

Metformin, which lowers circulating insulin levels and the ovarian production of androgens, has been found to reduce the follicular and estradiol response to stimulation and to increase the number of mature oocytes and embryo quality and the pregnancy rate in clomiphene-resistant women with PCO (15). In a subsequent study of unselected PCO women, the success rate was significantly increased only in normal weight women (16). As insulin resistance is more common in women who are clomiphene-resistant, that clinical group and insulin resistance may be particularly strong indications for this adjunctive treatment. PCO women on metformin who are coasted have lower peak estradiol levels and fewer days of coasting (17). As insulin is one of the main factors that stimulate vascular endothelial growth factor production by luteinized granulosa cells, and metformin decreases ovarian response and circulating insulin levels, metformin may be an important aid in reducing ovarian hyperstimulation syndrome in these women.


A semen analysis is done before the cycle to assure that semen quality is not at a nadir for that individual due to recent factors such as stress or a febrile illness. In general, IVF is preferred with reduced semen quality, as gamete intrafallopian transfer (GIFT) has been less successful than with normal sperm, and IVF allows confirmation of whether fertilization occurred. Pyos-permia can reduce sperm function (18). We attempt to clear pyospermia before proceeding to IVF. Frequent ejaculation may augment the action of antibiotics. Semen culture is probably worthwhile as a routine, in order to prevent the occasional contamination of the culture which will otherwise occur.


Cases of unexplained failure of fertilization have been found to be due to unrecognized subtle abnormalities of sperm structure. When strict morphology shows 4% or fewer normal sperm, the chance of failed fertilization is high. Insemination with a larger sperm number raises the fertilization rate to almost normal but the percentages of implantation and ongoing pregnancy/delivery are reduced by 40-50% (19), whereas intracytoplasmic sperm injection (ICSI) has been as successful as with other infertility factors (20). These findings suggest an embryotoxic effect of a high concentration of these very abnormal sperm which can be avoided by achieving fertilization with ICSI. In some cases, sperm morphology improves with observation or treatment with antioxidants. Sperm morphology may be impaired in smokers and may be improved by giving vitamin C, 1.0 g daily.


Antisperm antibodies (ASAs) in the female can impair or prevent fertilization if the female partner's serum is used in the insemination medium (21). Routine or selective use of fetal cord serum, human serum albumen, or donor serum will prevent this problem. As ASAs are also present in follicular fluid, we do extra washes of the cumulus and add an increased number of sperm. Although GIFT has been just as successful in women with as without ASAs (22), most women with high levels have probably been advised to have IVF. Female ASAs are more common when testing is done with her partner's sperm, suggesting antibody production to husband-specific antigens as well as non-specific sperm antigens. We currently test the husband's sperm against his wife's and against a negative control serum using the immunobead test.

Male ASAs may result from infection, trauma, or surgery, or may occur without any positive history. With greater than 70% IgG and IgA binding, there is a high chance of fertilization failure with routine insemination of the oocytes. ICSI is usually advised with high ASA levels.


It has been controversial whether the sperm penetration assay (SPA) is helpful, but one large study showed a very high predictive value of a 0% penetration rate with failed fertilization using a standard insemination number (23). Alternative methods of sperm preparation can improve both the SPA (24,25) and the fertilization rate (e.g., test yolk buffer and follicular fluid). If we have a couple who had their SPA done with test yolk buffer (TYB), we always use TYB for their IVF. Otherwise, one could have failed fertilization in an individual whose sperm only develop adequate capacity with TYB. As we have found consistent good results with the SPA with TYB, we now routinely use a 2-hr incubation with TYB for the IVF cycle but seldom do the SPA.


Fragmented DNA can be an unrecognized cause of infertility. This can now be determined clinically by flow cytometry sperm chromatin structure assay (SCSA). Although there is some correlation of abnormal sperm parameters with the SCSA (26), a high level of DNA fragmentation may occur with normal or mildly impaired morphology. In a recent study, antioxidant therapy was shown to improve the SCSA score. The impact of a high SCSA can be lessened by density centrifugation. A 450% improvement in nuclear integrity has been achieved with a 45-90% PureSperm® (Nidacon, Gothenburg, Sweden) gradient (26). Retrieval of testicular sperm may be an option for men with continuing high DNA fragmentation (27). In the same individuals, the level of fragmentation in testicular sperm averaged 5%, compared to 24% in the ejaculate. As there is a correlation with motility and morphology, choosing the most active and morphologically normal sperm for ICSI will also choose the sperm most likely to have intact DNA.


A number of reports have found a negative relationship of positive chlamy-dia antibodies to successful pregnancy (28,29). In one study, a significantly higher miscarriage rate was noted (30). This may be due to chronic endometrial infection or permanent effects of prior infection. Unfortunately, the endometrium can be positive with negative cervical cultures (31). In fact, in one study of 28 infertile couples with negative cultures or DNA probe assays, 40% were found to have active chlamydia infection by PCR. Because of these findings, we have elected to routinely treat both partners with a 10-day course of doxycycline. This may also eradicate ureaplasma and unrecognized semen or pelvic infections which could also compromise the outcome.


We have always done a rehearsal of the transfer with measurement and mapping of the endometrial canal. A controlled study has documented a significant increase in the pregnancy rate with this having been done with a reduced incidence of difficult transfers (32). It is helpful to do this under ultrasound guidance, in order to define the optimal conditions for embryo transfer. Cervical dilation has been shown to reduce the incidence of difficult transfers (33). Hysteroscopy has been used in very difficult cases to shave away ridges or cysts obstructing passage of the catheter (34).


The success rate with GIFT in women with tubal disease is not greater than with IVF, and the risk of tubal pregnancy is higher. Therefore, IVF is most appropriate with significant tubal abnormalities. We examine the uterine cavity with ultrasound before and during ovarian stimulation. Significant polyps or myomata are often easily visualized. A sono-hysterogram or hysteroscopy should be done if there is a further question of uterine disease. A recent randomized, controlled study has shown a higher pregnancy rate following hysteroscopic excision of small (mean 16 mm) polyps, underlining the importance of a thorough evaluation of the uterine cavity (35). Generally, a uterine septum should be incised before going on to IVF because of the higher risk of spontaneous abortion. Several recent studies have found approximately a 50% reduction in the rate of delivery in women with a hydrosalpinx compared with women with tubal disease without a hydrosalpinx (36). The success rate increases to normal after tubal repair or salpingectomy (37). Endometrial integrin is reduced in many patients with hydrosalpinx and reverts to a normal pattern after salpingectomy (38). Occlusion of the proximal tube seems to be equally efficacious (39). Spontaneous pregnancy can occur when a unilateral hydrosalpinx is removed (40) or repaired. It has been suggested that only hydrosalpinges which are visible on transvaginal ultrasound should be removed (41). However, hydro-salpinges enlarge during stimulation (42) and may become visible only during the IVF cycle. A recent randomized study showed increased fecundity following excision of polyps compared to only biopsy. Other studies have suggested that the polyp excision itself may enhance implantation from the healing process. A randomized study showed that a biopsy done in the cycle immediately preceding IVF was associated with increased implantation.


Most programs screen for HIV and hepatitis for safety of personnel. It would also be tragic to expend the amount of effort required to achieve an IVF pregnancy only to have the offspring at risk for a potentially fatal disease. With hepatitis B, the female partner should be immunized. With HIV, sperm separation and ICSI is being used by some programs to avoid transmission of the virus.


Some studies have shown reduced rates of implantation with severe or extensive endometriosis, and unexplained failure of fertilization has been reported in some women with endometriomas. A recent meta-analysis showed an odds ratio of successful pregnancy with IVF of 0.56 in women with endometriosis (43). Even in the presence of mild endometriosis, quantitative defects of the secretory response of endometrial glandular cells and other endometrial abnormalities have been described. Any endometrioma fluid should be kept separate from aspirates containing oocytes, and aspirating needles and pipettes should be changed. Two randomized studies have shown that a 3-6-mo course of GnRH agonist leading directly into IVF is associated with an increased pregnancy rate in women with stage III and IV endometriosis (44,45).


Viable pregnancy is reduced by about 50% with a history of DES exposure (46). Outcome is particularly poor with constrictions or a T-shaped cavity but is normal when the cavity is merely small.


Submucus fibroids markedly reduce the pregnancy rate with IVF (47). Studies have been conflicting regarding the role of intramural myomas, with some studies showing a significant reduction of outcome (47-49) and others not showing an effect (50-52). With relatively small studies, the statistical power is such that some studies may not detect a significant impact. It is likely that intramural myomas reduce implantation, but the effect is probably small unless the uterine cavity is distorted. Very large numbers would be required to accurately quantify such an effect. At the present time, we advise excision if they are large or distort the cavity.


Rarely, anxiety can lead to total inability to provide a semen specimen on the day of retrieval. Frozen husband's sperm has been found to yield a fairly normal rate of fertilization provided an increased number of sperm is added (53). In our detailed instructions to patients, we state in bold print: "If you anticipate any problems providing a semen specimen on the day of retrieval, please tell us. We can arrange for you to freeze a specimen as a back-up.'' A supply of Viagra should be available for any male having difficulty collecting a specimen.


Meta-analysis of studies on the effect of smoking on IVF conception rate revealed an odds ratio of 0.54 (95% CL 0.34-0.75) (54). Smoking also increases the rate of spontaneous abortion. We strongly recommend that all women stop smoking before having IVF.

A study of caffeine use found that intake of 2 mg (equivalent to one cup of decaf coffee) or less was associated with the highest pregnancy rate with IVF (55). Although not confirmed by other studies, avoiding caffeine is a simple measure to undertake.

Obesity correlates negatively with implantation (56). Therefore, weight loss may improve IVF results.

Studies on alcohol and fertility are conflicting, with some showing impaired fertility with small amounts of alcohol (57), whereas in one study, wine drinkers had a shorter time to conception (58).


Stress, anxiety, and depression have been linked to lower IVF outcomes (59-61), and psychological intervention improves the chance of success (62). Paying attention to these factors will also improve interactions of patients and staff, and will help adjustment to the stresses of child rearing. Multiple pregnancies have been shown to cause considerable personal and marital stress. Early intervention may enhance the long-term well-being of these families. Couples should plan their IVF cycle for a time of lowest possible stress.


Regular health screening such as pap smear or mammography can be easily forgotten during an extended course of fertility treatments. All appropriate health screening should be completed before embarking on pregnancy to avoid a significant health issue arising during pregnancy. For all egg donation recipients, we do a more extensive evaluation including a stress electrocardiogram, chemistry panel, and chest X-ray.


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