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Ultrasound in assisted conception

Ra/ar Goswam^
The use of ultrasound in the management of infertility is now rtnitine practice, not just as an aid in the diagnosisofovulaiion, but as an essential tool in the management of tile infertile couple. In the pre-ultrasound. era, ovulation monitoring was based on basal body temperature charts, changes in cervical mucus and serum progesterone checks. The use of ultr,lsou:^ti?in1£h'er:'.•!alT^.gCI7re^t;^'o&infertili^y.^was introduced by Krarochwil and colleagues' in 1972, when they first described the use of static imaging to visualize the Graafian follicle. However, it was not until the development o5- real-time ultrasound that the report of Hackeloer and associates7- in 1979 triggered renewed interest in this technique to track the growth of the Graafian follicle. This was followed by widespread use of real-time ultrasound to monitor ovulation in women undergoing donor Insemination and induction ofovuk-lion with clomiphene citrate3-4. The historical development of ultrasound techniques is beyond the scope of this chapter.. The author's airf. in this chapter is to demonstrate the use of ultrasound in the management of infertility, from the initial stages ofdragnosis of the cause of infertility, to the eventual confirmation of pregnancy, including routine monitoring of early pregnancy. Ultrasound research has further helped in the understanding of the physiological changes that occur during the ovarian cycle, especially with widespread use of Doppler technology in assisted conception. Guidance as to the specific advantages of ultrasound techniques and their usage should help those in the field of fertility management to utilize this technology to optimize benefits to their patients and to themselves. This chapter has been divided into four subsections, each of which is interrelated and not exclusive to the other.

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ULTRASOUND IN THE DIAGNOSIS OF-<i'N FERTILITY
Ultrasound? is probably the single, most important test wHen making a diagnosis, as to ch'e cause of infertility in a particular woman. A well-perrormed and derailed ultrasound examination of the female pelvic organs will give more information than any.oiher single test..

Methodology
First, ir must be emphasized that ultrasound examinations should be performed with the use of transvaginal probes. The use of abdominal ultrasound probes is outdated, i-.accurate and uncomfortable ro patients; it lacks derail and is indicated only when a large mass in the pelvis makes it difficult to visualize the uterus, ovaries and adncxal regions adequately. Second, although many examinations are performed 10 monitor follicle growth, mistakes are made and pathology is missed because a thorough systematic examinscion is not always carried out at each examination. It is important that a fixed routine is practiced during each ultrasound examination so that a systematic examination of the pelvic organs is carried out. The following routine is advisable. The ultrasound probe, lubricated with jelly, is inserted slowly, pointing anteriorly so as to locate the cervix and uterus. If the uterus is not seen anteriorly, a slow movement of the probe from side to side, and then pointing posteriorly, is made. Once the uterus has been located, it is important to note its position (anteverted or retroverred) and its size. Retroversion of the uterus may be physiological, but this is also more commonly noted in the presence of endometriosis. An enlarged uterus could be the result of pregnancy, adenomyosis or fibroids.

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In Vitro Fertilization and Assisted Reproduction

The uterus should be scanned in the longitudinal plane, so that the midline echo is located and followed from the internal os inferiorly to the rundus superiorly. The probe is then rotated so that a transverse scan of the uterus is performed. Once again, the midline echo is followed from one side of the uterine cavity to the other. Following this, the probe is rocked gently fri m side to side, to assess the adnexal regions. Any cystic areas, appearing echo free, can be assessed. This is especially important to distinguish hydrosalpinges from ovarian cysts (see below). After the adnexal region is assessed,' the ovaries are located on each side. By moving the probe laterally in the transverse plane, the ovaries can usually be located against the pelvic side-wall, medial or anterior to the internal iliac vessels. If the ovaries are not in the usual location, moving the probe anteriorly ard posteriorly will usually help with locating them. In some cases, especially in endometriosis, the ovaries may be anchored in the pouch of Douglas. Pointing the probe towards this area may make the examination uncomfortable for the patient, hence care should be taken that any movements are made gently and with attention to the patient's reaction. Both ovaries should be scanned sequentially, in the transverse and longitudinal planes. Before withdrawing the probe it is important to assess whether there are ai. esions present between the genital organs, the pouch ot iJouglas and the ovarian fossae. This can be assessed by placing one hand on the patient's abdomen and pushing towards the pelvis, first in the midline and then downwards from each iliac fossa. If there are significant adhesions, bowel loops will not slide against the posterior or anterior wall of the uterus. Similarly, restricted mobility of the ovaries against the pelvic side-walls may be indicative of adhesions in the ovarian fossae. To summarize, the ultrasound examination should be performed in a systematic manner. The examination is target orientated and a firm sequence should be adhered to at every examination. The simplest order of examination would be the uterus, adnexal region, ovaries and pouch of Douglas. The 'sliding organs sign' described by Timor-Tritsch and coworkers5 should become routine practice at all gynecological ultrasound examinations. Finally, uterine and tubal pathology can be missed, or physiological changes in the ovary may be wrongly diag

nosed as pathological, if the ultrasound examination is performed at an inappropriate time of the menstrual cycle.

Frequently asked questions (1) What information can be gained by a single ultrasound examination? (2) When is the best time in the menstrual cvcle to perform this examination? (3) What is the benefit to the patient? (4) What is the benefit to the clinician? Uterine factors The criteria for normal appearance of the endometrium and myometrium have been described previously 6"9. Uterine size and position and the endometrial cavity are studied in detail. The endometrial cavity and contours are inspected for irregularities and echo patterns and the myometrial— endometrial interphase examined in the longitudinal and transverse planes. The midline endometrial echo (Figure 1), where the anterior and posterior uterine walls are apposed to each other, are studied from the internal os to the fundus and any discontinuity and distortion of this echo should be noted. Longitudinal and oblique scans are performed to study the region of the tubal ostia.

Figure 1 Longitudinal scan of the uterus. Note the uninterrupted midline echo, indicative of a normal uterine cavity

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Large-intramural' 'and-^ubserous fibroids can distort the uterus. Especially in patients with polycystic ovarian disease. submucous fibroids are the most frequently missed' pathology in women described as having unexplained infertility. resulting in difficulties in ovum pickup because of abnormalities in the relationship between the ovary and the Fallopian tube. Fibroids can be diagnosed as well-defined hypoechoi1' areas arising from within the myometrial layer. synechiae or intrauterine adhesions should be suspected. This diagnosis can easily be missed if the ultrasound examination is performed when the Figure 2 Cornual fibroid distorting the right tubal region Figure 3 Submucous fibroid. Intrauterine septae are best diagnosed in the periovulatory phase. In cases in which endometrial thickness does nor increase synchronously with follicle development. The commonest abnormalities observed are fibroids. Intramural fibroids in the cornual region can affect tubal function (Figure 2).c^tion Several abnormalities in the endometrial cavity can be assessed on transvaginal sonography and many of these would be missed by digital vaginal examination. intrauterine adhesions. uterine septae and endometritis. Intrauterine adhesions cause interruptions in the midline echo (Figure 5) and are best noted in the periovulatory phase. polyps. The impact of fibroids on fertility is dependent on siB£and*locauon. They may result in abnormal thinning oftheendometrium in response to follicle development.the periovulatory phase (Figure 3)7 Endometrial polyps (Figure 4) appear as persistent hyperechogenic areas with variable cystic spaces. Uterine septae are best diagnosed in the transverse plane when myometrial echoes divide the fundal endo-meirial image (Figure 6). and these also distort the cavity contours. causing attenuation of the ultrasound beam and distal shadowing. Ac midline echo Figure 4 Endometrial polyp. However-. it distorts the midline echo and there is no distal shadowing 159 . It is hyperechogenic. Note the distal shadowing ind distortion o. Septae can usually be differentiated from intrauterine adhesions by the isoechoic nature of the septum with the surrounding myo-metrium.Ultrasound in assisted conc-. and ultrasound examination performed in a spontaneous cycle during the periovulatory phase. They are best seen mid-cycle and are not seen clearly in the mid-luteal phase or in stimulated cycles. endometrial thickness can increase quite markedly during stimulation for in vitro fertilization (IVF) and polyps can be misdiagnosed or over-diagnosed. although they can also be diagnosed in the luteal phase. Submucous fibroids distort the midline echo and are best diagnosed in .

' sonography findings as listed above were compared with hysieroscopy. Grainy. The sensitivity oftransvaginal sonography in detecting all abnormalities was 98.9% and the positive predictive value was 94. although most ultrasound workers agree that high echogenicity in the myometrium..cmum interphase appears diffuse and ill-defined (Figure 7).5% sensitivity for the diagnosis ofintrauterine adhesions. Fedele and colleagues" reported 100% sensitivity for transvaginal sonography compared with hystcroscopic findings in patients with submucous fibroids. abnormal transvafe.^^ ' / SlEMBtt ftbd & l5.7%.4%). Echogenic areas in posterior wall (asi isk) with thickening of posterior wall in a retroverted uterus . in a series of 193 patients. Adenomyosis is probably the most difficult diagnosis to prove. Figure 7 Endometritis. are indicative of adenomyosis (Figure 8). with loss of endometrial laye The Churchill Clinic »AOE. but should be considered as abnormal if there is a periovularory follicle present in the ovary.KDt1Y05IS ^. The presence of such an endometrial echo is quite normal in the luteal phase.. The positive predictive value for diagnosing submucous fibroids was 91.89!4B fc B4/B .IDS UB1BBM T6. Serial uluasound scans should be performed in at least two consecutive cycles to correlate increased echogenicity within the myometrium with endometrial echoes. midline echo almost invisible endometrium is extremely thin in the early fbllicular phase. Endometritis is diagnosed when the endometrial layers appear grainy in the periovulatory phase and the endome'rium —myoi'p.3%.TBrt MI:0.3 U: Figure 8 Adenomyosis. similar to endometrial findings. but 98. Thin endometrium. However.in Vitro Fertilization and Assisted Reproduction Figure 5 Intrauterine adhesions. which was similar to that in the diagnosis of polyps (91. our findings indicated only 91% sensitivity for fibroids. In a previous publication10.

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5 cm should be considered as abnormal. Note the thickened strona and peripheral distribution of follicles give a similar appearance to endometriosis on ultrasound examination and the differential diagnosis can often be difficult to make. Ovarian factors The unstimulated ovary. Cysts that are sharp and smooth in their outline and unilocular are usually physiological and do not warrant surgery16. the ovaries may contain ten or more cystic structures distributed peripherally around a central core of stroma (Figure 10). The benefit to the clinician is that operating list times can be adjusted accordingly and the right equipment made available for the patient. in premenopausal women.tic hysteroscopy can be combined with the therapeuric procedure.5 cm X 2. so mat the Other procedures can be carried out at the same time as the diagnostic procedure. In answer to the 'frequently asked questions listed above. Eshel and co-workers15 have shown that the presence of polycystic ovaries is more likelv to be associated with infertility if the woman has a raised body mass index. The presence of ovarian endometriosis may hav. containing solid and semi-solid areas.0 cm and ovaries with diameters in excess of 3. These are randomly dispersed and if the ultrasound examination is performed between day 6 and dav 14 of the menstrual cycle a leading follicle can be identified (Figure 9). In some women. one can conclude that if only one examination is to be performed then this should be performed in the periovulatory phase to assess and diagnose pathology in the uterine cavity accurately..:eption Ih reply ro"ttre •'frequently asked questions-^listed above. hirsutism. number-.small.offollidie. The benefit to the pstient is that hysteroscopic treatment can then be scheduled -it the appropriate time of the cycle. in many cases corpus luteum cysts will mimic pathological cysts of the ovaries. This is to avoid confusion with corpus luteum cysts. The mean volume of a normal ovary is 5.Ultrasound in assisted. A normal ovary usually measures 2. raised luteinizing hormone (LH) levels. of*oyo^. if only one ultrasound examination is co be performed. usually contains a.4 ml12..2 cm X 2. Cysts that contain low-level echoes. are usually indicative ofintraovarian endometriosis (Figure 12). then this should be performed n the 161 .f:^Iucrures-. Such ovaries have been termed polycystic and are usually associated with menstrual irreguhrity. which are characteristically irregular. Dermoid cysts can Figure 10 Polycystic ovary. carried out for IVF thenpy or intrauterine insemination17. Ovarian cysts are best diagnosed in the preovularory phase (Figure 11). Dermoid cysts usually contain hyperechogenic areas and some of these may cause distal shadowing.' w. which may be unilocular or multilocular. Figure 9 Normal ovarypRandom distribution-. a significant bearing on the ovarian response to stimi-iation drugs and may become obvious only during con:rolled ovarian hyperstimulation. anovulation and an increased incidence of miscarriage13'14. so that diigno-.

With endometriosis. The benefit to the patient is that unnecessary surgery can be avoided and in cases where pathological cysts are diagnosed these cysts can be treated laparo-scopically at th^ time of the diagnostic laparoscopy. whether laparoscopically or by laparotomy.0 mm per day'9-20. In cases of polycystic ovaries. if there is evidence of adhesions in the pouch of Douglas or the ovarian fossae. The benefit to the patient of accurate diagnosis is that these procedures can be performed at the same time as diagnostic laparoscopy and the risk of ovarian hyperstimularion syndrome and multiple pregnancies is decreased in cases of polycystic ovarian disease that has been treated with ovarian diathermy. depending on the size of the leading follicle. the first scan should be performed between day 10 and day 12 of the cycle and then. several small follicles may be seen in the early follicular phase.the diagnosis. There is poor blood flow. The benefit to the clinician is that. periovulatory phase. Ideally. then the clinician may decide to use preoperative medical therapy for 3 —6 months in order to decrease morbidity associated with laparoscopic surgery. in order to optimize the chances of diagnosing ovarian abnormalities accurately. However. laparoscopic ovarian diathermv may be performed at the time of the 162 . In many cases physiological cysts will subside spontaneously and do not require further surgery. This will avoid the risk of hyperstimularion syndrome and will increase the chances of spontaneous conception for these women. A luteal cyst is commonly seen and may decrease in size during the follicular phase. if the diagnosis of an ovarian cyst is made. and if oily fluid is aspirated Figure 11 Simple ovarian cyst. with no distal shadowing follicular rupture.of endometriosis or dermoid cysts can be made in advance of surgical procedures being scheduled. serial scans can be performed every 2 or 3 days. a repeat ultrasound scan should be performed approximately 8 or 12 weeks later. In natural cycles. Follicular rupture occurs between 18 mm and 28 mm during natural cycles. with an average growth rate of 1. In order to monitor follicular development and rupture. between day 6 and day 10 of the menstrual cycle. so it is most probably a follicular cyst then precautions during can be taken to this avoid is spillage performed intraperitoneally surgery.2 mm to 2. Note the echogeniciry. In cases where a dermoid cyst is suspected. MONITORING OF TREATMENT CYCLES Serial monitoring of follicular development is useful in both natural and stimulated ovarian cycles. the dominant follicle is selected between day 5 and day 7 and other follicles will gradually decrease as this follicle develops 18. A baseline scan should be performed early in the menstrual cycle to avoid misinterpreting pre-existing cystic structures with developing follicles.In Vitro Fertilization and Assisted Reproduction diagnostic laparoscopy. ultrasound fineneedle aspiration can be performed under anesthetic. where the ovaries are enlarged with diameters in excess of 3. It is essential that serial monitoring is carried out to determine normal growth of the Graafian follicle and to determine Figure 12 Intraovarian endomecriosis.5 cm.

Most centers use ultrasound alone. (5) The examination is used to check-the distribution of the follicles in the ovary.1 9^32-58 J// FR02EH . i. (3) The presence of ovarian cysts. if the cyst is below 3 cm in size... Monitoring of IVF cycles is essential in order to avoid the hyperstimulation syndrome and to assess the ovarian response during the treatment cycle. Ultrasound scans should then be performed on alternate days and the injection of human chorionic gonadotropih (hCG)'given to induce ovularion when the mean follicular diameter of the leading follicle is between 18 and 20 mm. will help.! tCHURCHILL CLINIC Figure 13 Hydrosalpinx.•-<•.5 cm. a second scan performed un day 8 or day 9 of the cycle. can affect follicular development and may warrant treatment prior to IVF. If the follicles are distributed around the periphery.J-. FRie - iI ! ^ !•• 'OR 45 | --• ••' I . this may be indicative of decreased ovarian reserve. In many cases. less than 2 cm in maximum diameter. .Ultrasound in assisted conception Ovulauon induction ancMntrauterine insemination Ovularion induction treatment may be carried out with clomiphene in combination with gonadotropins. It is now almost routine practice in IVF centers that hydrosalpinges are removed or sealed at the cornual end in order to avoid toxic effects of hydrosalpinx fluid on embryos and implantation22. However. (2) The presence ofhydrosalpinges (Figure 13) should be noted because this may also decrease the chances of implantation with IVF21. An initial scan performed on day 2 or 3 of the cycle prior to commencing ovularion induction is again advisable. will aid the ovarian response.-to establish the number of ovarian follicles. this may indicate an increased risk of ovarian hyperstimulation syndrome..e. ^ '^ " <•" . (4) When ovaries are enlarged to greater than 3. In these cases higher than usual doses of gonadotropins may be required in order to provoke an adequate ovarian response. .. -^. prior to oocyte recovery.'" . In these cases ovarian stimulation with gonadotropin should be started at a lower than normal dose and can be increased after 6 or 7 days of therapy. with the dominant follicle being observed in one or other ovary. or with gonadotropins on their own. if the ovaries are smaller than the normal size. Follicles should normally be randomly dispersed. The In vitro fertilization It is important that ultrasound examination is performed between day 10 and day 15 in a cycle preceding IVF. especially endometriosis (Figure 14). . and if the enlargement is due to endometriosis then the ovarian response may be compromised. the riskofhyperstimulation syndrome is increased. if necessary.••• 69:44. Ampullary folds of the mucosa appear as incomplete septae Figure 14 Intraovarian endometriosis 163 . - CL50 TX-03 g ^Gfc. aspiration of the cyst. for the following reasons: (1) The examination is used to check that the uterine cavity is normal. if performed prior to stimulation.er 'iffF858 e^-. The presence ofsubmucous fibroids ' will decrease the chances of conception with IVF20. to monitor the ovarian response and to time the hCG injection.

Subsequent ultrasound scans can be performed on a daily or alternate day basis.. Monitoring is performed as for a natural cycle. We demonstrated that uterine perfusion increases in response to rising estrogen levels during the follicular phase. depending on the ovarian response. In anovulatory women. 'n be demonstrated with the use ofDoppler ultrasound techniques23'24. and hence poor uterine blood flow was considered to be a cause of previous IVF failure. In patients in whom the initial blood flow was noted to be normal.In Vin'o Fertilization anil Assisted Reproduction first scan should be performed prior to stimulation for the reasons given above and the next scan should be performed approximately 6-8 days after stimulation has commenced. This confirms a caus Figure 15 Uterine artery Doppler waveform 164 . We also reported that. This is the stage at which implantation is most likely tc occur. clomiphene citrate or gonadotropins or a combination of both can be used in order to stimulate ovulation. and uterine blood flow was noted to improve in all the cases. In the second publication24 we performed Doppler studies of the uterine arteries (Figure 15) to study the uterine perfusion response in patients who had failed to conceive despite repeated multiple embryo replacements in IVF cycles. The injection of hCG is usually given when at least 2 or 3 leading follicles . These patients were studied in spontaneous ovarian cycles and. In 10% of cases the blood flow to the uterus did not improve despite estradiol valerate. then ultrasound scans muJ? be performed on a daily basis once the lead follicle reaches a mean follicular diameter of 16 mm. in conception cycles. The therapy used to improve uterine blood flow was estradiol valerate 1 mg tablets taken for 21 days starting on day 5 of each cycle. The mean age of the patients was 36. the pregnancy rate with subsequent IVF attempts was similar to that of the first attempt. the uterine blood flov response improved in cases of polycystic ovaries afte laparoscopic ovarian diathermy. re above 18 mm in size. as' for treatment of women receiving donated eggs. ovulation induction or wrauterine insemination. The improvement in pregnancy rates was attributed to improving uterine blood flow. DOPPLER ULTRASOUND In previous publications I have reported that the uterine response to endogenous hormonal changes in the spontaneous ovarian eye. despite normal endocrine changes and basal body temperature changes. Other studies25"28 found similar changes occurring in the uterine artery in the periovulatory phase. If urine or blood monitoring is not performed to monitor the LH surge. In these patients high-dose estrogens were used. uterine perfusion continues to increase in the mid-luteal phase in contrast to non-conception cycles and that there is a premenstrual decrease in perfusion as a result of falling progesterone levels. and embryos are usually replaced 2 or 3 days after ovulation. They also confirmed indices of high resistance to flow in the uterine arteries before ovulation and at the time of ovulation and decreased resistance to flow during the luteal phase. decreases in the preovulatory phase in response to the preovulatory estrogen fall and increases in the luteal phase in response to the combined effect of estrogen and progesterone. there was an inadequate uterine response in 50% of the patients recruited. Continuing research in this field indicates thai patients who have polycystic ovaries also have pool uterine perfusion in about 40% of cases. with the peak at the mid-luteal phase. for three cycles.9 years and there was a significant improvement in pregnancy rates after improving uterine blood How in those patients in whom blood flow was noted to be poor. In our ongoing study (unpublished data). • « Frozen embryo replacement In most centers frozen embryos are replaced' during a natural cycle.

Ultrasound in assisted conception and effect relationship. it is important to realize that the diagnosis of a pregnancy at this stage does not indicate viability. With the use of transvaginal ultrasound. Kratochwil and Eisenhuc36 first reported that pregnancy could be diagnosed as early as the first week after a missed period. Other reasons for poor uterine perfusion are related to previous surgery for ectopic pregnancy and age. but this was with the use of abdominal ultrasound and static imaging. Doppler ultrasound scans of the ovaries have yielded information regarding angiogenesis.. Increase in ovarian blood flow is related to angiogenesis. DIAGNOSIS AND MANAGEMENT OF EARLY PREGNANCY In 1967. or 4 weeks and 1 day (since the last menstrual period). selection of the dominant follicle and help with the differential diagnosis of follicular cysts (Figure 11) from corpus luteum cysts. 165 . Doppler studies of the uterus in spontaneous ovarian cycles have helped in identifying patients who are likely to conceive. This can be assessed reliably only after fetal heart motion has been demonstrated. Other workers30'31 confirmed this work in subsequent publications when they showed that the resistance to flow decreased in the ovarian stroma and in the corpus luteum from 4 days prior to ovularion until approximately 4 days prior to the onset of menstruation. mately 15—18 days after egg recovery or insemination. In this paper they described technical limitations due to patient obesity. There does not seem to be any practical' benefit from deriving these correlations. Numerous workers32"35 have studied blood velocities in the ovarian stroma during the periovulatory period. The use of Doppler studies in the timing of oocyte recovery is of little practical value. which starts early in the menstrual cycle when the dominant follicle or the dominant ovary has been selected. Lawson37 reported ultrasound criteria for the diagnosis of ectopic pregnancy. and it was not possible to obtain signals in 27% of the women studied. Their paper. More recently. However.cycles during cycles in which ovarian stimulation has been carried out using gonadotropins. and have been shown to be a possible cause for failure ofIVF treatments. described the use of ultrasound using static techniques to diagnose an incrauterine gestation sac. They found that the resis-tance-withintthcaiovarian vessels decreased throughout the menstrual cycle and peaked with the mid-luceal phase. because mature follicles will yield mature oocyies and mature follicles normally produce progesterone in large quantities prior to ovulation. The gradual increase in the ovarian blood flow is related to increasing estrogen levels and the dramatic increase that is noted to be preovulatory is most likely to be related to a sudden increase in intrafollicular progesterone levels. without time-consuming and inaccurate Doppler studies. The gestation sac is invariably located eccentrically within the uterine cavity. requiring a careful search of the adnexal area. attempts have been made to correlate increased blood flow with oocyte quality. An echo-free space in a central position is more likely to be a pseudosac and this should raise the suspicion of an ectopic pregnancy. The uterine cavity shows a thickened endometrium with a gestation sac seen as a 2-3-mm echo-free structure surrounded by a reflective trophoblasric ring. Most centers performing assisted conception techniques will perform a blood test to check P-hCG levels approxi. In 1978. Ovarian Doppler Taylor and colleagues originally obtained Doppler signals from ovarian arteries and reported this in 1985 29. They found that incrafollicular blood velocity started to increase approximately 1 day before follicular rupture and that the rise continued for at least 3 days after the formation of the corpus luteum. the equipment is not sensitive enough to differentiate conception from non-conceprion. A simple blood test to monitor progesterone levels after the leading follicles have achieved a main follicular diameter of 16 mm is a simple way of timing the hCG injection prior to oocyte recovery. it is possible to image a gestation sac in the uterus within 1 day of a missed period. first published in German. The practical value of these studies is still unproven. In pregnancies resulting from assisted conception treatment it is possible ro diagnose multiple pregnancy at this very early stage (Figure 16). It is a well established fact that progesterone levels in the peripheral serum start to rise prior to ovulation. However. In conclusion.

. The head of the embryo is just distinguishable from the body.. The coccyx is prominent and umbilical cord pulsations are easily visible.• i-'^Y''A -o (a e >/'. The crown—rump length is approximately 2 cm. stomach. This is the region where the placenta will develop. By the 9th week knees and elbows have become obvious and th.^ 13:S4. By 6 weeks cvsnc spaces become visible on one side of the gestation sac.Ba Figure 16 Twin pregnancy. The choroid plexus becomes visible with the formation of the falx cerebri (Figure 17).esis is complete. The finding of equal-sized yolk sac. The gestation sac is double the size of the yolk sac. By 5 weeks the yolk sac is demonstrable and measures between 3 and 4 mm. The embryo is now the same size as the yolk sz. but most of the organs Table 1 Chronological assessment of structures in early pregnancy Ultrasound finding. .'•• BPD •'i. The head appears co be larger than the body. By this stage.i . Unequal sacs may indicate impending spontaneous reduction Figure 17 Ten-wttk pregnancy. The need to measure these structures is discussed later.'•/ •• •/ 01= 4S.E . which remains at 5 mm for the rest of the first trimester. At 8 weeks embiyonic movements. is approximately 120 beats/ min and Doppler signals can be detected. discernible limb buds and development of the central nervous system are obvious landmarks of development. which can be recognized by the presence of heart motion within ic. twice the diameter of the yolk sac. Pregnancy is conventionally dated from the first day of the last menstrual period. By 7 weeks the embryo measures 7—8 mm in length and the yolk sac measures 5 mm in diameter.. .c. ai this stage. Gestation sac Yolk sac Cardiac pulsation Embryonic moven-.nts Head and limb buds Yolk sac and head . :. although the presence of normal-shaped structures corresponding to each other in size is enough to estimate gesiarional age to within a few days.. hearr motion cannot always be demonstrated. Note the falx cerebri anc choroid plexus 166 .. it is possible to demonstrate reral heart motion in 99% of cases. f . At 10 weeks dl-e yolk sac starts to disappear and the fetal heart. . lower limbs are seen with their legs crossed. The head contains a-i-ingle cerebral ventricle..B9 U» B6/B1/W / Early OS BEUt 65 OistStS single i*Uw5ci woo •("•>.HC •"••' •tc^ . assuming a 4-week menstrual cycle. Organoger. The embryo measures 1 cm. In real time it is sometimes possible to see blood flow in these spaces. At the site of the cord insertion. which may simulate a second yolk sac and must not be mistaken for a double-headed fstus. This appearance must be distinguished from an exomphalos. which has a diameter of 4—5 mm. A chronological assessment of structures seen with transvaginal sonography in early pregnancy is shown in Table 1.qual in size Falx cerebri Cord insertion Gestational age (weeks) The Churchill Clitic w^ ?^ •. and head is typical of this gestational age.FL» „'. USD iiw4d . a physiological umbilical herniarion of the gut may be seen. urinary bladder and kidneys may b'e seen.isaa 48.In-Vitro Fertilization and Assisted Reproduction By 5 weeks after insemination or ovulation. in> the abdomen. The heart rare.

Echogenic ring outside the uterine 167 . Iftthe'insertion. The incidence of spontaneous abortions varies between 20 and 50%. Another predictor of poor outcome that is seen by iransvaglnal. Early detection of ectopic pregnancy is crucial to reducing mortality and morbidity and this is even more important if future fertility is to be maintained. Although ovarian ectopic pregnancy is extremely rare. Bleeding at the placental site may occur as a result of defective implantation in the first instance or of decreased uterine perfusion. A combination of hormone assays and ultrasound examinations form the cornerstone for diagnosis. the contents must be scrutinized for the presence of a yolk sac. Once the gestation sac has been located. Transvaginal ultrasound is superior for imaging the implantation site. Ultrasound in assisted conception pregnancies than those seen with viable intrauterine pregnancies38"40. In most cases. but these do not improve the sensitivity. Therefore. progesterone is an additional useful marker. the embryo and heart motion. The definitive diagnosis of ectopic gestation depends on the identification of a gestation sac outside the uterine cavity. This is common when there is no evidence of retroimplantation bleeds and when there is no live embryo. in some cases when the Ectopic pregnancy Ectopic or extrauterine gestation is still one of the leading causes of maternal mortality in the UK and is one of the most difficult diagnoses in gynecological practice. especially in superovularion cycles when rings are seen within the ovary'. but not abdominal ultrasound. This appears as a highly reflective ring with an echo-free area* within-it-(Figure 18). In assisted conception. it is important that this is differentiated from corpus luteum cysts. order to diagnose bleeding at the implantation site. depending on the patients' age. positive assays ofp-hCG in the absence of an intrauterine pregnancy will form the basis of a presumptive diagnosis of ectopic pregnancy. Small lacunae or cystic spaces become visible at the implantation site as early as the 6th week. It is not always seen with ectopic pregnancies and its presence does not indicate an ectopic pregnancy unless there are blood clots within it. Several studies have reported thai blood levels of hCG are lower in ectopic Figure 18 cavity Ectopic pregnancy.(Figure 19). By 12 weeks the physiological umbilical hernia has started to reduce and disappear. however. Fluid in the cul-de-sac is generally an unhelpful sign. In 50% of cases with bleeding noted in the placental site the pregnancy may still continue. Otherwise. a low progesterone level and a low(3-hCG level are indications for early transvaginal ultrasound examination41. Laparoscopic surgery is the cornerstone of treatment for ectopic pregnancy. Compressed by a full bladder. Serum progesterone levels are significantly lower on day 15 after oocyte recovery with ecropic gestation than in intrauterine pregnancies.are difficult to distinguish until they have grown to larger than 2-3 mm in size. is the presence of large vascular spaces surrounding the whole gestational sac. as with missed abortions. Only when embryonic structures are seen is the diagnosis of ectopic pregnancy certain. Color Doppler ultrasound can be used to look for increased flow patterns around the placental site. However. With the development of biochemical assays and imaging techniques it is possible to diagnose most cctopic pregnancies preoperatively. It is important that anatomical landmarks are used as a way of assessing fetal growth and that the placental site is examined at each ultrasound examination in. Color flow imaging and spectral Doppler to diagnose ecropic pregnancy have been proposed. there is no explanation noted for these cases of threatened abortion.of the cord is close to the site of placental bleeding the prognosis of the pregnancy is extremely poor. they may not be visualized by transabdominal ultrasound.

Bianchi S. Poison D\V. Franks S. 1988:15-25 6. London: Heinemann Medical Books. Thompson IE. AdamsJ. Dodson MG. J Clin Ultrasound 1990. is only the first step in its use. High-resolution cndovaginal ulrrasonography of the endometrium: a noninvasive test for cndometrial adequacy.6l:211-14 2.Vitro Fertilization and Assisted Reproduction Finally. et al. Narayan R.49:956-60 168 . Poison DW. after in vitro fertilization therapy lube is inaccessible. Lancet 1988. Dorta M.8: 239-42 9. Its potential as a diagnostic aid in threatened abortion and the speed with which a diagnosis of non-viability can be made make it a necessity for all assisted conception units.135:122-8 3. 78:200-4 10. Am] Obstet Gynecol 1982. Robinson HP. Abdulwahid NA. potassium chloride and methotrexate injections. Friedrich F.36:573-6 4. ct al. in a patient. BrMed J 1988:297:1024-6 15. Armar NA. Fertil Steril 1981. ACKNOWLEDGEMENTS With grateful thanks to Hayley Bridgeman for her patience in preparing this manuscript. The best agent for injection is yet to be established. Gordon AN. Eshel A. Rottem S. Grunfield L. conception and early pregnancy loss in polycystic ovary syndrome. Prevalence of polycystic ovaries in women with anovulation and idiopathic ligutism. ultrasound has become an essential part of the management of infertile couples. 144:569-73 5. The uterus: a new look vuth transvaginal sonography. CONCLUSION In conclusion. ultrasounddirected injection of methotrexate may^be applied. Elgali S. Lewit N. Walker B. eds. Influence of serum luieinizing hormone concentrations on ovulation. Deter RL. Timor-Tritsch IE. Polycystic ovaries -a common finding in normal women. Bergh PA. Krarochwil A. Goswamy RK. et al. for laparoscopic surgery. Adams J. / Clin Ultrasound 1990. et al. Transvaginal ultra-sonography versus hysceroscopy in the diagnosis of uterine submucus myomas. Its accuracy in the imaging of the uterus and intrauterine abnormalities makes it imperative that the diagnosis of unexplained infertility is never made without a systematic ultrasound examination being performed at the appropriate time in the menstrual cycle. ctal. Am] Obs'. Eshel A. Definition of anatomical planes for use in transvaginal sonography. saline. The role of ultrasound in ovulation induction — a critical appraisal. Rottem S. Kletzki DA. References 1. Siebel MM. Wadswonh J. Urban GV. Pulsatile luteinizing hormone-releasing hormone therapy in women with polycystic ovary syndrome. In Timor-Tritsch IE. Varygas JM. Fedele L.In.3: 129-33 11. BrMedJ 1986.J Clin Ultrasound 1990. Ultrasound Obstet Gynecol 1993.293:355-9 14. 18:337-49 7. Transvaginal sonography of the uterine cavity with hysteroscopic correlation in the investigation of infertility. Rottem S. Correlation of ovarian follicle size and serum estradiol levels on ovulaiory patients following clomiphene citrate for in vitro fertilization. Obstet Gynecol 1991. ct at. Fertil Steril 1988. How transvaginal sonography is done. etal. Hackeloer BJ.j. Transvaginal Sonography.77:745-8 12. ct al. Fleming R. Ultrasonic tomography of the ovaries. Figure 19 Corpus luteum rings. Ann Chir Gynecol 1972. Follicle monitoring. Correlation of ultrasonic and cndocrinologic assessment of human follicular development. results so far being similar with hyperosmolar glucose. Marrs RP. Armar N. Transvaginal scanning of wdomm'ium. Entman SS. Fleischer AC.1:870-2 13. Obstet Gynecol 1991. including the timing of the posccoital test. McArdle CR. Thaler I.18:331-6 8. transvaginal ultrasound has replaced abdominal ultrasound in early pregnancy monitoring and diagnosis of ectopic pregnancy. Homburg R.et Gynecol 1979.

cycle: a transvaginal Doppler study. Santolaya-Forgas J. Romero R. Intrafollicular blood flow during human ovulation. et al. Goswamy RK. Packe TD. Ultrasound Obstet Gynecol 1992. Impaired implantation after in vitro fertilisation treatment associated with hydrosalpinx. Growth patterns of non-dominant ovarian follicles during the normal menstrual cycle.2:197-202 31. Fertil Steril 1993. Ultrasound dopp-ler flow studies of (he ovarian and uterine arteries. endocrine function and intra-follicular blood flow during the periovulatory period. Pampiglione JS. Edinburgh.33:272-6 . et al. Coy RA. Bourne T. Ovarian morphology. Uterine and ovarian perfusion during periovularory period assessed by transvaginal color Doppler. etal. Geburtshiife Frauenheikd 1967. In Vitro Fertil Embryo Transfer 1989:6:338-41 18. et al. Ultrasound in the diagnosis and treatment of ovarian cystic tumours. et at. Hum Reprod 1990. 38. 27:176 37. Merce LT. Eisenhut L. Treatment ofsubmucus fibroids. Gynecol Endocrinol 1990:4:245-50 26.SongIO.AdoniA. Williams G. Kurjak A. Schulman H. Dervain I. Br Med Obstet Gynaecol 1985. it al. Chorionic gonadotrophin and progesterone levels in ectopic pregnancy. Effect of hydrosalpinx and its surgical correction on pregnancy and implantation rates following IVF and embryo transfer. MaclerJ. Transvaginal color blood flow imaging of the peri-ovulatory follicle. Bourne TH. / Ultrasound Mod 1992. Hum Reprod 1988. etal.W 21. Fertil Steril 1990. etal. Fertil Steril 1993. Fertil Steril 1989:52: 981-5 Ultrasound in assisted conception 28. UK. Goswamy RK. Am J Obstet Gynecol 1988. Garces D. Renaud RL. Doppler ultrasound studies of the uterine anery in spontaneous ovarian cycles. The effect of endometriomas on in vitro fertilisation outcome. Obstet Gynecol 1986.131:153-8. 11:139-42 27.158:1239 41. DeJon. dysovulatory and anovulatory cycles. et al. etal./:•<•rt»7Afr^7 1980. et al. Decreased . Stepcoe PC. Obstet Gynecol 1977.1994. Iniraovarian Doppler velocimetry in ovularory. Narayan R. Diagnosis of ectopic pregnancy. Wells PNT.54:638-42 19. Pittaway D. Scholtes MCW. Lawson TL.47:722 40. Kadar N. Jurkovic D. Presented at the 13th Annual Meeting of the European Society for Human Reproduction and Embryology. ' 50:1945 -V r' 169 . Stepioc PC. 193:268-72 22. Serial human chorionic gonadatrophin measurements in ectopic pregnancy. Wikland M.60:433-8 35. CollinsW.* 20. Ectopic pregnancy.6:319-24 33. Kurjak A. Ultrasound Obstet Gynecol 1991. Am JRoentgenol 1978. Hum Reprod 1991. Campbell S. / Am Assoc Gynaecol Laparosc.a cause of infertility. Goswamy RK. HCG determinations in early pregnancy. ScgalS. 12 (abstrl):P-lll 23. et al. Waterstone J. Hum Reprod 1991. and outcome of assisted conception. Br J Obstet Gynaecol 1996. Watersrone J.16. Larocca E. Romero R. Campbcll S. MilwidskyA. Echographic study of follicular maturation and ovulation during the normal menstrual cycle. Kupesik-Urek S. Transvaginal color flow imaging of the uterine arteries during the ovarian and menstrua! cycles.60:439^3 36. 1:307. uterine perfusion . Fleming C. KoongMK. Barco MJ. Taylor KJW.56:870-3 32.1:53-9 34. Kadar N. van Rijen HJM.Jurkovic D. Hum Reprod 1997.68:440 39. Der fruheste nachweis der fecalen herzakrion durch ukraschall.92:240-6 30. Dieterle S. Bourne TH.6:721-6 24. et al. Steer CV. WladimiroffJW. DIugi AM.5:391-5 29. Banaglia C. Kratochwil A. Fertil Steril 1987. Burns PN. Hum Reprod 1988. Granberg S. Uterine and ovarian flow velocity in the normal menstrual. Transvaginal color flow Doppler in the assessment of ovarian and uterine blood flow in infertile women. Lanzani A.6: 177-85 17. Wladimiroff JW. Son IP. Fertil Steril 1991. criteria and accuracy of ultrasonic diagnosis. Doppler ultrasound Studies of the uterine arteries in spontaneous and IVF stimulated ovarian cycles. Hull MGR. Physiology of the menstrual cycle by ulirasonography. Kupesic S.8: 955-9 25.

The need to retrieve individual human oocytes by a less invasive procedure. The first culture of human oocytes in vitro from ovarian tissue removed at laparotomy is ascribed to Gregory Pincus some 25 years before the same experiments were repeated by Robert Edwards'. gamete intrafallopian transfer (GIFT). PRELIMINARY ASSESSMENT It should go without saying that a complete infertility assessment should always have been carried out upon all couples who are about to embark upon assisted conception treatment. The need for preliminary laparoscopy Prior to the advent of ultrasound-guided oocyte recovery. either laparoscopically or transcervically. there are many couples who do receive treatment by assisted conception who have been inadequately investigated. This chapter describes the methods of oocyte recovery and embryo transfer that have evolved over the past 20 years and how they are now practiced at Bourn Hall. oocyte recovery via the vagina using vaginal scanners became possible6. Lancashire. The only recent developments that have occurred are those involving the transfer of embryos. a consultant gynecologist in Oldham. It was for this reason that Edwards. the scientist. From then. since the retrievals could be performed with sedation only or light general anesthesia. and Steptoe. intrauterine insemination (IUI) and other techniques is described in detail elsewhere (Chapter 2). 171 . with the potential of aspirating oocytes from them 2. was recognized early on by Edwards. the gynecologist. Unlike oocyre recovery. laparoscopy was the only technique available. Ultrasound-guided retrieval also meant thai all patients could be treated as day cases. particularly with regard to real-time scanning. The proper assessment of couples prior to IVF. However. it is appropriate to discuss briefly the place of assessment or preliminary laparoscopy in the management of patients. Thus. accessibility of the ovaries for oocyte recovery. these are described in Chapter 17. particularly with regard to the . nevertheless. the technique for embryo transfer (ET) has hardly changed since the first successful transfer in 19783. until by 1981 it was shown to be possible consistently to retrieve oocytes by needling follicles with transabdominal ultrasound guidance5. it was essential that the current state of the pelvic organs was known. Oocytes were collected for in vitro fertilization (IVF) exclusively by laparoscopy from then until the great technological advances made in ultrasound imaging allowed ovarian follicles to be clearly seen 4. as the technology further improved and with further miniaturization of transducers.Oocyte recovery and embryo transfer 11 techniques for in vitro fertilization Peter R. He came to hear of Patrick Steptoe. who in 1968 was the only man in England operating with the laparoscope. Systems were continuously improved. methods of oocyte recovery became increasingly simple.N-TRO. to the Fallopian tube. came together to collaborate and ultimately to create the first child born as a result of in vitro fertilization and embryo transfer (IVF-ET)3. He was therefore able to see the female pelvis in a minimally invasive way and to see ovarian follicles. Initially for research and later for the treatment of infertility. It was. Embryos are still transferred at Bourn Hall in exactly the same way as was taught by Steptoe.DUGTLON. Then. Brinsden I. less traumatic to the patients and simpler for their surgeon.

9 procedure. There are. It was a furthe 2 years before a live birth was achieved. normal practice to carry out. however. we usually collect the oocytes by transvaginal ultrasound-directed recovery. However. This preliminary laparoscopy allowed one: (1) To plan the route of entry of the collecting needles. unless all the embryos are frozen in that cycle for future transfer. Ultrasounddirected oocyte recovery has revolutionized the way IVF is carried out. This is also because the majority of patients have been fully assessed before being referred for treatment at Bourn Hall. (2) To divide adhesions in the pelvis. Laparoscopic oocyte recovery As early as 1968 Patrick Steptoe devised a method ol aspirating oocytes laparoscopically from human pre-ovulatory follicles (Figure 1). especially if the tubes are known to be blocked. laparoscope and trochar. a-laparoscopic assessment on every patient who had not had one in the previous 6—12 months7-8. (2) When it is considered desirable to assess the state of the pelvis at the same time as collecting oocyies. if GIFT or zygole intrafallopian transfer (ZIFT) is proposed. Each of the major oocyte recovery techniques is now described. it will not be possible to assess the Fallopian tubes. infection or endometriosis. However. (3) To decide whether in fact a laparoromy was required to separate adhesions and to bring the ovaries into full view. At present. then the presence of adhesions is of little consequence. (5) To record in exact detail any abnormal findings.In Vitro Fertilization and Assisted Reproduction therefore. the first ever IVF pregnancy. in order to save a second procedure. on the rare occasions that GIFT is carried out.. then it is essential that a recent assessment of the tubes should have been made. (3) If it is intended to make an attempt at GIFT. particularly around the ovaries. (4) To occlude Fallopian tubes that were patent but abnormal. This was especially important when there was extensive adhe-ion formation. assessment laparoscopies are now required less frequently. we do not consider that preliminary laparoscopy is necessary if it has been done within a reasonable time. still three remaining indications for laparoscopic oocyte recovery: (1) When suitable ultrasound facilities and experience with ultrasound-directed oocyte recovery are not available. to allow future access. In 1976 a pregnancy wa: achieved following IVF of oocytes from laparoscopil oocyte recovery. which wa: subsequently found to be ectopic9. or unless the state of the pelvis is likely to have changed for the worse because of recent major surgery. it has meant that IV!' can be practiced without anesthesia and as an outpatient Figure 1 Mr Patrick Steptoe performing a l<tparoscopic oocy recovery in the 1970s 172 . LaparoscopL OOCYTE RECOVERY TECHNIQUES The change from laparoscopic oocyte recover}' to ultrasounddirected oocyte recovery is now almost universal. and not laparoscopically. so as to prevent the occurrence ofecropic pregnancies following embryo transfer. If standard IVF treatment with ultrasound-directed oocyte recovery is the selected treatment. and then proceed to the laparoscopy. If this is done. This should seldom be the case in reputable units. Because IVF is now invariably carried out with ultrasounddirected oocyte recovery at Bourn Hall and because GIFT and ZIFT are rarely used treatment options.

bleeding and difficulty in recovering oocytes. in order to prevent postoperative chest and shoulder pains.flushed through with flushing medium (Earle's medium with heparin). The clear follicular fluid is seen to enter the oollecring tube. these may be gently divided by blunt dissection or by the use oflaparoscopic scissors. rather than through its thin dome. where the tissue is thicker.the follicle may be gently curetted as the vacuum is app-ied. we have used the Cook pump (Cook (UK) Ltd. since this may cause rupture of the follicle and loss of the contents. noti»h. are aspirated.. the laparoscope. which can be maneuvered into a suitable position for oocyte recovery by grasping and manipulating the ovarian ligament or the inftindibulopelvic ligament (Figure 4). however. Every effort should be made to remove all the remaning gas. can nake laparoscopic oocyte recovery a most rewarding procedure.. a procedure of the pasc.gas-rinsufflaror. methods for oocyte recovery and IVF.»fluidt Tha-lafcoiaiorv. the suction pressure of which can be controlled by a foot switch. A 1cm transverse subumbilical incision is made and a Verre's needle is inserted through the abdominal wall and directed towards the concavity of the pelvis. the follicle is "flushed with an equal volume of flusiing medium and aspirated until it is identified. UK) (Figure 3). Full general anesthesia is induced. Often the oocyte is found in the second or third flush. which has subsequently been confirmed11'12. When the ovary is in a suitable position and the follicles are clearly seen. occasionally an oocyte may be found which has dropped into the pouch. it would certainly still be possible to do so. 5% CO^ and 90% N2. the CO. which. The ercbry-ologisr will be able to determine the maturity of the follicle by the appearance of the granulosa and curr-Lilus cells.«and«tthe^presenceo^viscous. Semms. The selection of patients. There have been no developments-over the past 10 years that would improve upon the method originally described.delivers-a. and more recently. but a ncunber of problems can arise which can turn it into a fmstrsdng and prolonged procedure. The follicle to be aspirated is best maintained uppermost and punctured from the side through an avascular area (Figure 4b).afc>Bourn Hall since 1988. ultrasound imaging of die pelvis ' has been the greatest advance in the diagnosis and 173 .'When-an adequate pneumoperitoneum has been achieved. flowofCO^ at'a-pressure of'less'tnan-100 mmHg. We used the Craft pump (Rocket of London. the needle and tubing. steady.are descriBed in detail in Chapter 12. On completion of the procedure. Ultrasound-guided follicular aspiration techniques Apart from laparoscopy. If minor adhesions obscure access to the ovaries. even the small ones. allows a better seal around the needle to be obtained. ovulation induction and monitoring are described elsewhere in this book.. Laparoscopic oocyte recovery is a straightforward oro-cedure for an experienced laparoscopisr. their investigation. grasping forceps and needle are withdrawn and th. At the completion of the recovery the pouch of Douglas should be aspirated clear of all blood and flushing £uid. A fine trochar and cannula are passed into the peritoneal cavity from a lateral position and a double-lumen follicular aspiration needle is passed down it. Puncture from the side. A good view of the pelvic organs is essential. gas mixture is released. having been attached by fine plastic tubing to the collection tube or pot and to a vacuum pump. endometriosis.. this has always been the practice at Bourn Hall. collection may begin.or Palmer-type grasping forceps are inserted in the midline or laterally about 2. as 100% COa causes acidification of follicular fluid8'10. but for most of us. If difficulty is experienced in obtaining the oocyte. placed in the Trendelenberg and lithotomy position as for' any laparoscopy and the urinary bladder is emptied.eeni»practiced. The needle is inserted into the follicle and a vacuum is applied up to a pressure of 100—110 mrrHe..5 cm above the symphysis pubis or inguinal ligament (Figure 2). The major problems arc the unexpected finding of adhesions obscuring the ovary... The needle is connected^to. are. All visible follicles. Patients are prepared. with increasing experience and expertise. a. Before aspiration. The peritoneal cavity may be flushed through with 100% CO. which is then also released.Oocyte recovery and embryo transfer cechn'^nci oocyte recovery-has. UK) originally. is changed to a gas mixture of 5% O. draped. The first clear aspirate is then passed through to the embryologist to detect the oocyte.. Proper selection and preparation of patients. Letchworth. particularly of the ovaries. If it is not found.

(d) Cook (K-FTH-1012) te tube hearer (Cook (UK) Ltd.. UK). (b) close-up view to sh the position of the instruments. Letchworch. UK) 174 . Letchworth. (c) Cook K-MAR-4000 follicle flushing system (Cook (UK) Ltd.. and we : now have the ability to measure blood How to the ut< and ovaries with color Doppler imaging. we have progressed to real-time images on vaginal scanning which can even detect the minute cumul oocyte complexes in mature ovarian follicles. (Reproduced with kind permission of Professor lan Craft) Figure 3 (a) Craft vacuum pump (Rocket of London. (a) General view showing the position of the operator and assistants. UK). Figure 2 Laparoscopic oocyte recovery.. UK). Letchworth. (b) Cook K-MAR-5100 vacuum pump with foot controller (Cook (UI1 Ltd.In Vitro Fertilization and Assisted Reproduction management of gynecological conditions. From the earliest pioneering days of lan Donald in Glasgow and crude A-scan and B-scan static images of fetuses in utero.

with aspiration needle entering ovary ai an angle.t ttansvaginak ukrasoundsdirected oocyte recovery technique. This was followed by the first recorded aspiration of.ccQtf6r•gencral*3.'. At present.b\yrhc. other units were practicing ultrasound-directed oocyte recovery successfully14 and it was found that recovery rates were as good as by laparoscopy. and gave as good oocyte recovery races and fertilization rates as laparoscopic retrieval15'18. (b) right ovarian ligament grasped by forceps to steady ovary. (c) as (b) but with right infundibulopdvic ligament grasped Methods Transabdominal—transvesical ultrasound-directed oocyte recovery (Figure 5a and b) may be performed under general anesthesia.l•'. proximity of the transducer to the ovaries. Transabdominal—transvesical ultrasound-directed oocyte recovery This was the original method described by Lenz and colleagues 5 for the recovery of oocytes from ovarian follicles. The technique was originally described as using a fixed needle guide attached to the abdominal transducer. ease of learning. Shortly thereafter. but with abdominal ultrasound to guide the aspjration-of follicles16'17. but its great advantage over 175 . Figure 4 (a) General view of pelvis showing normal uterus and Fallopian tubes with stimulated ovaries.cslhcsia•a. but others have found that this limited the flexibility of approach and have found a 'freehand' method without the guide to Be preteraBle19'. as practiced at Bourn Hall. In the following sections. the very large majority ofIVF units collect oocyres.\4. patient acceptance •.Oocyte recovery and embryo transfer techniques It was following the pioneering work of Kratochwi! ano coll&gues-'v'wiTO developed •rh'c uirrasonically guided transcutaneous needle biopsy techniques of abdominal organs. with fewer complications and easier ovarian access'5 (Figure 5).d•relati^• elyfew complications. Soon. oocytes were successfully collected by the transvaginal route.J••o'-'-«»•l:i^c^i!'. This approach has very significant advantages: simplicity of use.oocytes from follicles under ultrasound guidance by Lenz and colleagues in 19815. followed by a detailed description of the transvaginal recovery technique. the ultrasounddirected oocyte recover}' techniques that evolved before the transvaginai technique are described. It was used by most IVF units up until it was superseded by the vaginal ultrasound method as the method of choice. that observations on ovarian follicular development "were made13.

In Vitro Fertilization and Assisted Reproduction .

SP. (e) transvaginal oocyte recovery using a vaginal ultrasound probe with attached needle guide. F. follicle. (d) trans-abdominal-perurcthral oocyle recovery. AW. (b) trans-abdominal-transvesical oocyte recovery (freehand technique). TV. vagina. ovary. T. (c) transabdominal-transvaginal oocyte recovery. abdominal wall. needle 176 . U. (a) Transabdominal— transvescial oocyte recovery (fixed technique). symphysis pubis. N.different ultrasound-guided oocytc recovery techniques. uterus. V. B.Figure 5 Diagrammatic representations of. transducer. bladder.

When the follicles have been identified. 177 . The surgeon may maneuver the ultrasound transducer personally. The abdominal probe is placed in a sterile polythene sleeve containing contact gel and the pelvis is scanned to define the uterus and ovaries. UK) 10 mg intravenously provides excellent sedatio. with full aseptic technique. Pressures of 100120 mmHg mav be applied. Roche Produces Ltd.-» anpisra-n'dX'T.wirh-srerile-warer-and» sterile abdominal drapes are applied. but with the freehand technique it is preferable to hsA'e alhexperienccdi. which is then aspirated (Figure 7). controlled with a foot pedal. Most pumps have a 'red button' or second foot control for higher vacuum pressures to clear the needle and tubing of blood clot or tissue.icaacsa-Si. The bladder is carhecenzed and emptied of urine. which becomes visible as a point of increased echogenicity if one is in the right plane. At the end of the procedure the flushing medium and blood that has accumulated in the pouch of Douglas are aspirated.. and on to a suction pump.'T. Alternatively. the follicle is flushed with a volume of flushing medium (Earle's solution with heparin) equal to that aspirated.Oocyte recovery and embryo transfer techniques laparoscopy is that sedaawfaloncrcair b*e''used?' T+i'isican be achieved with premedicarion 1 h before the procedure: intravenous diazepam. nocmafc* saline OR SSrtnTatB!. followed by Dellenbach and co-workers17 who also later presented-the results of a large series of patients20.». The operation may be carried out in a 'clean' procedure room. su^uio^. If the oocyte is not in the first aspirate. and flushed and aspirated until the oocyte is obtained or until the embryologlst indicates that there is little prospect of recovering one. Its disadvantage is that it is quite painful for a number of patients.i with almost complete amnesia following the experience. The other major problem is the relative distance of the ovaries from the ultrasound probe. (Reproduced with kind permission "of Professor lan Craft) to use the operating theater. Discussion Transabdominal—rransvesical ultrasound-directed oocyte recovery was a very considerable advance technically over laparoscopy for the recovery ofoocytes. we have found that midazolam (Hypnovel. which involves the use of a transabdominal ultrasound probe with needle aspiration of the ovarian follicles through the vagina (Figure 5c). a small bleb ofl% lignocaine is raised in the skin. The collecting needle is attached by tubing to a sterile plastic collecting tube. 500-m! bai^ of<. Transabdominal-transvaginal ultrasound-directed oocyte recovery This method. Welwyn Garden City. was first des cribed by Gleicher and colleagues16. is thrust firmly into the most accessible follicle. to a. UK) and washed off.hebladder .5—10 mg* and' pethidine 25-100 mg are given slowly intravenously intraoper-atively. ultrasonographer to help.:h'os»i'lrl'e'd" Trie sodomen is prepared with chlorhexidine (Savlodil. Quite severe hematuria is not infrequent. making accurate needling of the follicles more difficult. but we prefer Figure 6 Transabdominal-transvesical freehand technique. This technique is now very seldom used and has entirely been superseded by the vaginal approach. ICI Pharmaceuticals UK. with occasional clot retention of urine post-operatively. followed by a small nick with a scalpel close to the transducer in order to ease the passage of the needle through the skin. particularly because the needle passes through the sensitive bladder wall. Wilmslow. The tip of the needle. and an indwelling self-retaining catheter is inserted and connecred. The same procedure is repeated for all the follicles on each side. A double-lumen needle is passed down along the track of the ultrasound beam 'freehand' (Figure 6) through the abdominal wall and the distended bladder towards the selected ovary. We have used a number of different ultrasound machines and abdominal probes and have found that they have all performed adequately.

Transvaginal ultrasound-directed oocyte recovery It was not until 1985 that vaginal ultrasound probes became commercially available and their usefulness for oocyte recovery immediately became apparent. They presented data on a series of 242 cases 22 with no serious complications. (c) and (d) the needle tip in a follicle This method is seldom used now because of the advent of vaginal ultrasound probes. at which time they also described the method of guiding the collecting 178 .. Wikland and colleagues6 first suggested the potential of vaginal ultrasound for oocyte recovery in 1985. or if a vaginal probe is out of order. or if it has malfunctioned during a procedure. the direct abdominal approach will often solve the problem in these cases. then this technique would have been much more widely used. It will continue to be used in units that do not have vaginal probes. Very occasionally. or in the presence of vaginal infection. as the ovaries are much closer to the end of the probe.In Vitro Fertilization and Assisted Reproduction Figure 7 Ultrasound picii. oftransabdomlnal—transvesical oocyte recovery. which have made the vaginal approach very much more accurate. The only indication now for the use of this method is if a unit does not have a vaginal probe. It may also be used if the ovaries are inaccessible vaginally. If vaginal transducer technology had not advanced so rapidly. ovaries are situated high in the pelvis and are difficult to access with a vaginal probe. (a) The needle entering the bladder. Transabdominal-perurethral ultrasound-directed oocyte recovery Parsons and colleagues21 first described the method of collection of oocytes via the urethra and bladder using transabdominal ultrasound probes (Figure 5d). (b) the needle entering the bladder wall.

either by. The echo from the tip of the needle. the follicle is flushed with.in. almost every IVF unit collects oocytes by the transvaginal ultrasound-directed method. The follicular fluid passes into plastic tubes in a •hoc block' or Test Tube Heater (Cook (UK) Ltd. It is usually possible to keep the needle tip within the ovary and aspirate several follicles. hand or by means of an abdominal pressure cufp3^ the mobility of the ovaries can be reduced and entry into the follicles made easier. the quality of the endo-metrium. One of the follicles nearest to the probe is selected. In this way it is often possible to aspirate all the follicles from both ovaries with Figure 8 Ultrasound picture of transvaginal oocyte recovery with the echogenic tip of the needle visible in a follicle 179 . Lerchworth.an equal volume of warmed flushing medium. The full tubes are passed to the embryologist in the adjacent laboratory. Now. UK) is attached to the ultrasound probe. the vaginal transducer is introduced into the vagina and the pelvis is scanned thoroughly to confirm the position of the uterus. UK).'fl^^-aColfereri^ either a light general anesthetic or analgesia. the vulva and vagina are prepared with Savlodil and rinsed thoroughly with normal saline. warmed normal saline.5 MHz) head is currently used at Bourn Hall.under. The vaginal transducer is covered with a sterile polythene sleeve or special 'condom'.. with a small amount of coupling gel to cover the end of the probe. UK) with £ variable two-frequency (5 and 7. Ovaries are often mobile and'tend to 'run away' from the tip of the needle. If analgesia alone is requesredxiheiw^s'-ad'mirasTercdfes. transvaginal ultrasound-directed oocyte recover is-jyrformed. UK) with a 7-MHz vaginal mechanical sector transducer or a Sonoline SL1 machine (Siemens pic.Oocyte recovery and embryo transfer techniques needle alongside th^-traasdnGcrsto-acHrcvc'very accurate puncture of follicles (Figure 5e). can be seen in the follicle. which is inserted into the vagina and directed towards the posterolatera'l aspect of the vaginal fornix. since it is the easiest. either Earle's medium with heparin or. Although some recommend that the needle be withdrawn and flushed after each oocyte has been recovered24. and the si^sroWDSG&wr^s^seTitedESjtc&Ps^. The follicle is then aspirated^ again and this is repeated until the oocyte is recovered. and the oocytes are identified. the most accurate and the most acceptable to patients of all the collection methods. Gentle manipulation of the needle tip within the follicle may assist release. The patient is placed in the. we believe that the number of stabs through the vaginal vault and ovaries should be kept to an absolute minimum. London. the-operating. The needle guide (Casmed. Methods * At Bourn Hall.rheater. in order to reduce trauma and the possibility of bleeding. If an oocyte "is not found in the first aspirate. Lecchworth..of oocytes chat-are reluctanMo separate. fullaseptic technique. By using a snort-stabbing movement and by applying pressure supra-pybically. UK) double-lumen needle is inserted partwav into the needle guide. A PIVET-Cook (Cook (UK) Ltd. which is scored to increase its echogenicity. its maximum diameter is found and the needle is then thrust gently but purposefully into it (Figure 8). the position and accessibility of the ovaries as well as the number of follicles to be aspirated. A Phillips SDR 1500 machine (Phillips Medical Systems. which is then aspirated until em^ty.. by careful manipulation and by choosing the order in which they are aspirated.'Qcsceffetecstb'oye'f&r*-transabdominal-transvesical ultrasound-directed oocyte recovery. Initially. as we have been using more recently. Sunbury-on-Thames. one after the other.sl-i'arryth'is is assucedt* by using single-use disposable needles.. Cheam. This effect can be reduced by ensuring that the needle ti(y are ket^t verv. the majority choose general anesthesia.lithotomy position. and the ultrasound probe is then manipulated and rotated until the follicles appear in close proximity to the needle guide lines.

UK) 1. 1981 1982. oocytes. because there is no need to cathetel the bladder or pass the aspirating needle through [here is much less chance of dysuria. inal vault is checked with a speculum for signs of bleeding. occasionally it may be necessary to insert an absorbable suture at the site of bleeding. so was vaginal ultrasonography. thus necessitating less analgesia and sedation. the pouch of Douglas is aspirated of any visible pool of Hushing fluid and blood. beause of its closer proximity to the ovaries. reference 25) and updated to 1989 (A. This shows the Scandinavian experience of the change from laparoscopic to transvaginal ultrasound-directed oocyte recovery between 1981 and 198925. and very effectively. Alternatively. Sunde. (5) The fact that.. because it is painful than ultrasound-directed or laparosco oocyte recovery. 180 . inecdon hematuria than after transabdominal-transvesi ultrasound-directed oocyte recovery. because the bladder wall is not pierced. (2) Greater ease of use and shorter learning phase. The majority ofIVF units throughout the world now do their oocyte recoveries by transvaginal ultrasound guidance. been reported. UK) 1 g rcctally at the end of the procedure. (6) Less risk of perforation of a viscus because of proximity of the ovaries to the vaginal vault. it will invariably stop with firm pressure for a minute or two using a sponge in a sponge forceps. or an intravenous pyelogram-cype c will help to stabilize the ovaries. with a consequently quicker return to normal activity. may be obtained from smaller follicles. because of the better visualization of the ovaries and of the smaller follicles.In Vitro Fertilization and Assisted Reproduction a single puncture on each side. We aspirate all follicles that are visible. both imraoperatively and post ranvely. How the 'fashion' changed in the use of different method. more oocytes are recovered. When all follicles are seen to have been emptied.. during which time the same very major change in our own practice occurred. we give prophylactic antibiotics during the procedure in the form ofAugmentin (Smith-Kline Beecham. If bleeding does occur.2 g intravenously. Eastbourne. (3) Less pain. At Bourn Hall.'f oocyte recovery as described in this chapter is demon&iiated in Figure 9. As the use of laparoscopy was extended to oocyte recovery. There are a number of measures that can be taken ensure that the transvaginal method can be made ea and safer: (1) Pressure on the lower abdomen provided b' lumbar-type corset with foam pads placed su{ pubically. The major advantages of transvaginal over transabdominal-transvesical ultrasound-directed oocyte recovry are: (1) The better image obtained with the vaginal probe. even those of less than 10 mm in diameter. The transducer is then withdrawn and the va. in general. personal cc munication) Discussion Vaginal ultrasound has undoubtedly been the greatest advance in our ability to visualize the pelvic organs since laparoscopy. (4) The fact that. the publishers of Human Reproa tion. (7) Much better patient acceptance. as mature oocytes. (2) A double-lumen needle with flushing and aspirai channels achieves more efficient and rapid retriev. and Flagyl (RPR Ltd. Brentford. assistant can be asked to press firmly on the lo abdomen with a pad when difficulty with a mo ovary is encountered. which can be fertilized. more embryos are available transfer and freeze and therefore higher pregai rates have. 1983 1964 1985 1986 1987 * " Laparoscopic ~ Transvesical — Transvaginal Figure 9 The Scandinavian experience of the changes in 'fashi of oocyte recovery techniques between 1980 and 1989 (coun of Oxford University Press.

The patient is placed in the lithotomy position and the surgeon places a perineal drape over her and inserts a Cuscoe vaginal speculum lubricated with warmed saline solution. immediately posroperarively. In the laboratory. The length of the uterine cavity will usually have been measured before the treatment cycle. Brentford. At this stage the lights in the theater are kept dimmed. If difficulty is experienced in passing the catheter through the cervical canal. The catheter is fitted with a 1-ml tuberculin-type syringe and flushed through with medium. Nor do we keep patients lying down for 12 h after transfer. especially those who are felt to be particularly at risk for multiple implantation. so that a volume of 15-25 P. We no longer place patients in the knee-chest position when transferring to an antevened uterus. The catheter is taken through to the theater and passed to the surgeon.there being a belief that the results are improved by doing this. Those embryos that are to be transferred are placed into a drop ofEarle's medium. scrubbing up and gloving. At Bourn Hall. an Edwards-Wallace (Bourn) embryo transfer catheter (Smiths Industries Medical Systems (SIMS). UK) rectally. embryo transfer techniques (3) It is essential to maintain the sharpness of the needles. but are switched up v/hen the surgeon is ready to do the transfer. The embryo(s) are drawn up into the already charged catheter. There are occasions when an apparently 'goodlooking follicle' turns out to be the internal iliac vein in cross-section! Rotation of the probe will show it to be a long tubular structure. The cervix is exposed and any vaginal and cervical secretions are gently removed with small pledgets of cotton wool moistened in normal saline. the embryos are identified by the embryologist and scored. not yet been a controlled trial to prove this theory. The embryos are usually at the 2-4cell stage of cleavage by then. UK) is used for the majority of transfers (Figure 10).1 is transferred.i. it-is always wise to look at them in two planes by rotating the transducer before entering them. Lancing.e. and their details are entered into the log. transfer is carried out about 48-50 h after oocyte collection. EMBRYO TRANSFER The basic technique of transferring embryos to the uterus following IVF has changed very little at Bourn Hall over the past 10 years. the identity of the patient is checked by the accompanying nurse. RPR. Embryo transfer is carried out in the operating theater under sterile conditions. which is about 44—48 h after insemination of the oocytes. a gentle approach will tend just to push the ovary away from the needle. but not gowning up. In the UK. (4) The number of entries of the needle through the vault of the vagina as well as the ovary should be minimized. then the stiffer outer sheath 181 . Husbands are encouraged to attend. It will often be possible to aspirati all follicles from one ovary with one stab only. If there is a plug of thick mucus in the cervical canal. A firm and rapid thrusting motion should be used to penetrate the follicle. as was originally recommended by Steptoe (personal communication) and by Jones and colleagues26. UK) intra-operatively and metronidazole (Flagyl. We have moved towards transferring only two embryos in the majority of women. so that the tip of the catheter can be placed about 1 cm from the fundus.Oocyte recovery and. Eastbourne. the Human Fertilisation and Embryology Act (1990) (see Chapter 33 for more detail on this legislation) made a rule that a maximum of three embryos may be transferred. • Methods (5') When-scannihg-follicles. this may be wiped away or aspirated gently. the surgeon and the embryologist attending to the case . On arrival in the theater. there has. SmithKline Beecham. but otherwise the procedure is practiced much as it always has been. Chapter 12 gives more detail on the laboratory aspects of the transfer procedure.all then sign the case records confirming that they have done so. under the age of 36 and previously fertile. however. More recently we have been placing embryos rather lower in the uterine cavity — at a depth of 5—6 cm from the external cervical os . The catheter is gently maneuvered through the cervical canal and into the uterus. suitably gowned and with overshoes. Generally. (6) \Ve advocate the routine administration of antibiotics in the form of clavulanic acid (Augmentin.

If this fails. and after a maximum of 2 min (because of the cooling effect on the embryos).In Vitro Fertilization and Assisted Reproduction can be introduced into the canal and the inner catheter 'persuaded' through. the outer sheath is held in position and the stiffener withdrawn. If it is still found to be impossible to pass the catheter after every reasonable means has been tried. the speculum is withdrawn and the patient is made comfortable. When the operator is confident that the catheter is properly placed. then the embryos are returned to the laboratory and replaced in the culture dish. The embryo transfer catheter is removed from the outer sheath and replaced by the stiffener which. the embryologist will draw up the embryo(s) agaiiq into the catheter and a further attempt will be made to transfer them to the uterus. In earlier. patients are transferred to the ward in a wheelchair after the transfer' and allowed to lie flat in bed for a half an hour. and currently at Bourn Hall. the catheter is left in position for a few moments and then gently and slowly removed. The Wallace catheter now comes with an optional stiffener for the outer sheath. can be molded to a shape that may facilitate navigation of the cervical canal. 182 . the embryologist or the surgeon can slowly and gently inject the embryos into the uterus. it is threaded up the sheath and will usually enter the uterine cavity. days the patient remained resting with a slight head-down tilt to her bed for 2h before| being allowed home. There is no evidence that this is necessary. or transferred to a comfortable reclining armchair with a footrest. an Aliss single-tooth forceps may be applied 10 the anterior or posterior lip of the cervix and gentle traction applied to straighten the cervical canal. because it has a 'memory'. If they have. The embryologist then returns with the embryo transfer catheter. The catheter is returned to the laboratory and checked to ensure that the embryos have not been retained. If the internal os is negotiated. On completion of the transfer.

bladder bleeding 40%. Birth after reimplantation of a human embryo. or Utrogescan (Besins Iscovesco. References 1. Lureal phase support is given to all patients who have been on analog protocols.If it still proves impossible to transfer the embryos because the catheter cannot be passed into the uterus. occasionally.C. fiberoptic light sources and video imaging. 400mg twice or three times a day. We use either Cyclogest (Shir. The simplicity of the procedure is its great merit. Isiedn. Pharmaceuticals Ltd. intramuscular progesterone in the form of Gesrone (Ferring Pharmaceuticals Ltd. With the development of smaller and smaller ultrasound transducers. France) 100 mg twice -or three times a day. attempts were made to move closer to the ovaries by perurethral and transvaginal routes. we believe that transvaginal ultrasound-directed oocyte recovery is a very much safer procedure and will remain the method of choice for oocyte recovery long into the future. The dramatic improvements that occurred in ultrasound equipment in these same two decades enabled us to monitor closely even small changes in follicular size and endometrial texture. UK) vaginal pessaries. In spite of the administration of prophylactic antibiotics intraoperatively.2:366 183 . Paris. Lancet 1968:2:913 3. but it is our experience that complications are very much less frequent than were reported by Discussion Since the earliest reports ofoocyre recovery from human ovaries by laparoscopy . There are no other recent reports with as much detail as these. all of which resolve with intravenous antibiotic therapy. There are an average of one or two pelvic infections each year. vaginal bleeding 24%. safer. with venous puncture 2— 7%. then the procedure is abandoned and arrangements are made for the transfer to take place the following day under general anesthesia. Laparo-scopic oocyce recovery techniques have changed little in the past 20 years except for refinements in instrumentation such as laparoscopes. A Matter of Life. Lancet 1978. postoperative pain 10% and pelvic infection 3%. Vaginal ultrasound has brought a new dimension to the diagnosis and management of gynecological disorders. Edwards RG. Wlkland and colleagues24 reported no serious complications in 50 cases. UK) 50-100 mg daily is used.. vaginal probes have given us the ability to view the pelvic organs in great detail..well. as it is easier for surgeons to learn. The development of ultrasound-guided intra-abdominal biopsy techniques in the early 1980s enabled fertility specialists 10 'biopsy' ovarian follicles and aspirate oocytes from them5. Andover. particularly endometrioric cysts. requires no anesthesia and has much greater patient acceptance than laparoscopic and transvesical ultrasound-guided oocyte recovery. bowel perforation 14%.tolerated. the aspiration of cysts. Laparoscopy and ovularion. 1980:40 2. Brinsmead and colleagues28 reported relatively high complication rates. unless indicated for therapeutic reasons. and has certainly revolutionized the practice of oocyre recover}'. Sicptoe PC. In the past 5'years at Bourn Hall there have been no admissions to hospital for bleeding following oocyte recovery. These infections have frequently been associated with aspiration ofendometriotic cysts. It was then only a 27 Brinsmead and colleagues. From there. London: Hutchinson. Human chorionic gonadotropin is never used for luteal phase support. Langley. if the vaginal preparations are" not.Steproe P. Edwards R. several major changes in technique and technology have simplified the procedure and made it much more acceptable to patients. When compared with the incidence of complications occurring at laparoscopic oocyte recovery. Oocyte recovery and embryo transfer techniques short step to using guides mounted upon these probes to enable very accurate localization and' aspiration of ovarian follicles to be carried out. appears to be better avoided. this has been with an average of 900— 1000 oocyte recovery procedures each year. The incidence of complications for transvaginal ultrasound-directed oocyte recovery is variously reported. inserted per vaginam. Steptoe P. The Story of a Medical STcakthrough.

Current status ofin. Edwards RG. Hansmann M. Transvesical and transvaginal approaches for the aspiration of follicles by the use of ultrasound. Hamberger L. Dellenbach P. A Manual of Gamete Handling Procedures.39:241-3 27. Nilsson L. 1st edn. Grudzinskas G. Lancet 1981. Veek L. Edwards RG. GarciaJE. Moreau L. Siegel I. Friberg J. Stepcoe PC. Webster J.6:149-54 /' 184 . Lancet 1984. Tokyo: Springer-Verlag. New York. Brinsmead M. Edwards RG. Hamberger L. Campbell S.442:683.2: 508-9 17. Establishing full term pregnancies using cleaving human embryos in vitro. Ultrasonic tomography of the ovaries. 1:244-9 16. Riddle A. London: Helnemann. Steptoc PC. PampiglioncJS. Urban G. Riddle AF. Transvaginal sonographically controlled ovarian follicle puncture for egg retrieval. Plumere C. Oliver M. Follicular fluid pH changes following ini raperitoneal exposure ofGraafian follicles to carbon dioxide: a comparative Study with follicles exposed to ultrasound. Lcnz S. Wikland M. Wikland MD. Lancet 1985. IVF in the Nordic Countries 1981-1987: a collaborative survey.15:245-51 25. Collection of human oocyics by the use of sonography. Steptoe PC. Fullan N. Yovich J. Laparoscopy for oocyic recovery. Oocyte retrieval for invitro fertilization by ultrasonically guided needle aspiration via the urethra. FertilSteril 1983. Daya S. Devroey P. Collection of human oocytcs for in vitro fertilization by ultrasonically guided follicular puncture. Fertil Steril 1985. 1:683-9 28. Kahn JA. Biological Basis and Clinical Application. Lancet 1976. Parsons J. Vitro Fcrtil Embryo Transfer 1984. Transvaginal sonographically controlled follicle puncture for oocyte retrieval. Kjellow M. Fer:il Steril 1987:48:454-8 20. Freidrich F. 1983: 83-94 14. Ultrasound diagnosis of follicular growth and ovulation. Hum Reprod 1988. Mayden K. JIn Vitro Fertil Embryo Transfer 1989. 1990:127 24. Sandow BA.442:178-81 9. Wikland M. Verbessem D. Enk L. 3:727-30 12. PurdyJM. Egg retrieval for in vitro fertilization by sonographically controlled culdocentesis. Raymonds. Hum Reprod 1988. Laparoscopic or ultrasonically guided follicular aspiration for in vitro fertilization?//?. Rlumere C. Pampiglione JS. Sunde A. Pneumoperitoneum induced pH changes in follicular and Douglas fluids during laparoscopic oocyte recovery in humans. Ann NYAcadSci 1985. Ann Chir Gynaecol Fenn 1972.!: 880-2 10. AkkermannsJ. von During V. Booker M. Wilson L.3:751-4 13. Feger B. dark L. Sieptoe PC. Steptoe PC. Kesky T. The Management of Infertility. Sharma V.1:213-16 19. Two years' experience of ultrasound-directed oocyte retrieval. J din Ultrasound 1987. Rumplcr Y. Mason BA. Nisand I. Morcau L. 1:1467 18. Van Steirteghem A.603-8 15. Wikland M. Gerlinger P. Dellenbach P. Brun B. Lancet 1970. ShumackJ. Hackeloer Bj. Laurirsen JG. 44:656-62 21. FertilSteril 1990. Gerlingcr P. Nilsson L. Fcgcr B. Hum ^roa'1990. Glelcher M. Ultrasound as a diagnostic and operative tool for in vitro fertilization and embryo replacement (IVF/ER) programs.9 7. Enk L. Feitchinger W.39. Camu F.vitro fertilization and implantation of human embryos: Lanctti 1983:2: 1265-9 11. eds.5:959-64 26. Giglia RV. Acosca AA. Mambcrger L.6l:211-14 5.1:1076 22.53:97-102 23. Lindner HR. Molne K. Reimplantation of a human embryo with subsequent tubal pregnancy. A randomised trial of laparoscopy and transvaginal ultrasounddirected oocyte pickup for in vitro fertilization. Kratochwil A. Whitehead M.In Vitro Fertilization and Assisted Reproduction 4. Laparoscopic recovery of preovulatory human oocytes after priming of ovaries with gonadotrophins. Campbell S. Campbell S. Ann NYAcadSci 1985. / In Vitro Fertil Embryo 7M?H/CT-1984. Goswamy R. Jenson PO. Parsons J.1:1163-4 6. Heidelberg. Stangerj. Jones H\V. Hansson R. Use of a vaginal transducer for oocyte retrieval in an IVF/ET program. Edwards RG. Nisand I. Hammarberg K. Lancet 1983. Parsons J. Ultrasound directed follicle aspiration for oocyte collection using the perurethral technique. Fertil 5^n7l983. Kcmeter P.87:737 8. Ford NT. Br J Obstet Gynaecol 1980. On (he transfer of conceptuses from oocyies fertilised in vitro. In Beier HM. Berlin. SharmaV. Fertilization of the Human Egg In Vitro.