The following chapter is called "Follow-ups with a Positive or a Negative Pregnancy Test".

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1 Slide 1 Welcome to chapter 7. The following chapter is called "Follow-ups with a Positive or a Negative Pregnancy Test". The author is Professor Pasquale Patrizio.

2 Slide 2 This chapter has the following objectives: How to establish follow-ups for patients with either a positive or negative pregnancy test; What to rule out in patients with a positive pregnancy test. In particular the risks for multiple pregnancies, ectopic pregnancy and the definition of biochemical pregnancy and the meaning of first trimester bleeding will be reviewed; What to do for patients with repetitive implantation failure.

3 Slide 3 The first serum assessment for pregnancy is obtained on the 15th day from oocyte retrieval (in practice this will be 12 days after a day 3 embryo transfer and 10 days after a day 5 embryo transfer). It is impossible to predict a pregnancy outcome from the first beta hcg result. However, levels below 20 IU are rarely associated with a good outcome. If the beta hcg does not rise properly (at least 66% higher than the previous one), it is important to consider the possibility of a biochemical pregnancy, (defined as a pregnancy documented only by blood test without evidence of a gestational sac), or an ectopic pregnancy. For the calculation of the gestational age, the first day of the last menstrual period is by convention two weeks prior to oocyte retrieval. The first ultrasound (US) is ordered when the beta hcg level is around 2,000 IU (the current ultrasound resolution power allows first visualization of pregnancy) to document pregnancy in utero and presence and size of the yolk sac (about 5 weeks of gestational age). A week or two later a second ultrasound will document the final number of gestational sac(s) and the presence of embryo pole with heart beat.

4 Slide 4 The ultrasound exam at 6 weeks documents clearly the presence of the embryo pole (indicated by the arrow) on the side of the yolk sac.

5 Slide 5 Twenty-eight days after embryo transfer (about 6 weeks from last menstrual period (LMP)) the fetal heart activity can be verified for the first time. This slide shows the presence of the embryo pole measuring 3.0 mm on the side of the yolk sac. Fetal heart activity was noticed. If the next ultrasound 1-2 weeks later shows that the embryo is growing properly and the fetal heart activity is confirmed, the patient can be told that her chance of delivery is about 90% (Rosen GF, Silva PD, Patrizio P et al. Predicting pregnancy outcome by the observation of a gestational sac or of early fetal cardiac motion with transvaginal ultrasonography. Fertil.Steril.54(2):260-64, 1990).

6 Slide 6 A third ultrasound is performed at around 9 weeks of gestational age and if everything is progressing normally, the patient is instructed to continue progesterone supplementation up to 10 weeks and to make appointment for obstetric care. In USA patients are provided with instructions to inform the reproductive center of the final outcome of the pregnancy so that these data can be reported to the Society of Assisted Reproductive Technology (SART).

7 Slide 7 This slide reports the definitions used to identify the various types of pregnancy. Biochemical pregnancy is a pregnancy that can be documented only by a positive beta hcg test on a blood sample; since these pregnancies represent a very early arrest of the embryo growth they are not visualized by ultrasound. A blighted ovum pregnancy is defined by the presence of a gestational sac visible with ultrasound, but no identification of the embryo pole within it. A clinical pregnancy is defined by the presence of a gestational sac with yolk sac, embryo pole and heart beat on ultrasound. A missed abortion is any loss of a clinical pregnancy prior to 20 weeks of gestation.

8 Slide 8 An ectopic pregnancy is defined as a pregnancy implanted in an abnormal location and it can be further subdivided into: tubal pregnancy (the most common type) whereby the pregnancy can be located in any segment of the fallopian tube, although the ampullary portion is the most common tubal location; ovarian or abdominal are ectopic pregnancies located on the ovary or in the abdomen respectively; and cervical are those ectopic pregnancies implanted in the cervix of the uterus. Heterotopic pregnancies are defined by the simultaneous presence of a gestational sac within the uterus and a second one (rarely even a third one) in any of the ectopic locations.

9 Slide 9 In this case at 6 and half weeks of gestation the ultrasound does not show presence of embryo pole. Only a yolk sac is visible. This pregnancy had to be classified as abnormal. Four days later there still was no presence of an embryo pole. Therefore, a missed abortion was diagnosed.

10 Slide 10 The procedure on how to terminate an abnormal pregnancy should be chosen based on the dialogue between physician and patient. Medical and expectant management may take a long time and the patient may experience heavy and sudden, unpredictable vaginal bleeding. Dilation and curettage (D+C) provides a rapid resolution in order to resume a subsequent cycle and provides the opportunity to obtain fetal tissue for genetic evaluation. Medical options include misoprostol - an oral or vaginal prostaglandin tablet or oral progesterone antagonists. With misoprostol a common regimen is 600 µg per os every 6 hours. It can be repeated up to 3 times in 24 hour period. Once bleeding and cramping starts the patient can discontinue the medication. If the medical treatment fails then D+C becomes necessary. Finally, it is important to follow the decline of Beta-hCG to undetectable values.

11 Slide 11 First trimester bleeding may occur in up to 20% of pregnancies. In about 50-60% of these cases, even if persisting for the entire first trimester, they do not cause a pregnancy loss. The main reasons are either subchorionic or retroplacental hemorrhage, suggesting a suboptimal implantation process. The therapeutic options are limited to observation with bed rest, to provide reassurance and to check the administration of progesterone.

12 Slide 12 This slide shows the many possible locations for ectopic pregnancies. The most common sites for ectopic pregnancies are the ampullar and the isthmic portion of the fallopian tube, while least common locations are the ovarian site, the abdomen and the intramural site of the uterus.

13 Slide 13 The therapeutic options for the various types of ectopic pregnancies are summarized in this slide: The cervical ectopic pregnancy can be treated by ultrasound-guided injection of KCl in the gestational sac or, if present, in the fetal heart; sometimes it may be necessary to perform also a suction D+C. For cornual and abdominal or ovarian ectopic pregnancies the only option is surgery. Tubal pregnancies can be treated with medical intervention by using methotrexate if there are no contraindications, or surgically with salpingostomy or salpingectomy. Heterotopic pregnancies are very rare and almost always require surgery; however it is often possible to preserve the uterine pregnancy (except in cases of cornual and intrauterine pregnancy). A heterotopic pregnancy must be promptly recognized as the ectopic pregnancy can rupture and because of massive blood loss requiring emergent surgical intervention.

14 Slide 14 The risk of adnexal torsion is increased in patients undergoing ART. During ovarian stimulation treatment, patients develop multiple large follicles/cysts that predispose the ovaries to twist on their blood vessels. Adnexal torsions are difficult to diagnose. Most patients feel generally uncomfortable and an ultrasound with doppler does not always lead to a conclusive diagnosis. However, when ovarian torsion is strongly suspected, an immediate laparoscopy is needed to both diagnose and untwist the adnexa after draining the multiple ovarian cysts.

15 Slide 15 On this slide you can see the three most common questions asked by patients after a negative pregnancy test: a) Why did the treatment fail? b) What is the next step? and c) Is it time to stop and seek alternative options? Most of the time there are no definitive answers and an initial approach might state that human reproduction is very inefficient. Two recent studies (Kovalevsky G and Patrizio P, 2005; and Patrizio P and Sakkas D, 2009) showed that only 15% of the embryos transferred and only 5-6% of all the oocytes retrieved result in a live birth. Next, it is important to do a full and detailed review of the failed cycle.

16 Slide 16 A detailed review of the failed cycle should include: The type of ovarian stimulation protocol used. For example, if the ovarian response was suboptimal (few follicles/oocytes) the dosage of medication should be increased at the next cycle. If there was an asynchronous growth of follicles and the estradiol levels were also indicative of suboptimal follicular recruitment by not raising accordingly, the next protocol should be changed. It is important to review the number and quality of the oocytes. If there were fewer oocytes than expected (according to the number of follicles) and if their quality was poor (for example many oocytes displayed vacuoles or polar body fragmentations or excessive cytoplasm granularity) the protocol should be changed. If the same phenomenon is observed again at the repeated cycle, it is likely that the reason of infertility is related to the production of poor quality oocytes and other alternative options should be discussed. If there were many immature oocytes (for example 30-40% of metaphase I or germinal vesicles), the timing of administering hcg at the subsequent cycle should be extended (perhaps one or two more days of stimulation). If there was poor fertilization in conventional IVF, the next cycle should offer ICSI (intracytoplasmic sperm injection). If the embryo quality was poor (for example embryos that failed to cleave or showing early developmental arrest or excessive fragmentation) the protocol should be changed. It is also important to check whether poor embryo quality was observed in more than one patient. In this latter case a review of the embryology laboratory performance is warranted. If the embryo cleavage arrests after two days of in vitro

17 culture, it has been suggested to anticipate the day of the embryo transfer (for example from day 3 to day 2). If the embryo transfer was traumatic or difficult, every effort has to be directed towards improving this extremely important step (as discussed in the chapter on embryo transfer techniques). If the patient has extra embryos frozen, she should be encouraged to use these embryos since it is likely that the competent embryo (i.e. the one destined to result in a live birth) may be in this cohort.

18 Slide 17 In about 10% of patients multiple IVF treatments, despite the production of good quality embryos, do not lead to a successful outcome. These patients are classified as having recurrent implantation failure (RIF), defined as no pregnancy after 3 or more embryo transfers (of which at least two with fresh embryos), in the same center and with embryos of good to excellent quality. The great majority of patients with RIF are diagnosed with unexplained infertility.

19 Slide 18 The post-treatment consultation with couples with RIF is difficult because there are no explainable reasons for the recurrent failures. It is important to review the entire clinical history and to consider additional tests such as karyotype on both partners and thrombophilia screening (even if there is no clear evidence to support it). If a laparoscopy was never performed it might be indicated (to reveal occult endometriosis). It is worthy to consider changing the treatment protocol (even if the follicular response was optimal). It is also useful to consider assisted hatching in a subsequent cycle or to switch the day of the embryo transfer (postponing the embryo transfer to cycle day 5, blastocyst stage to have the opportunity to observe the embryo growth).

20 Slide 19 In some instances preimplantation genetic screening may help to explain the failure (for example all the embryos have aneuploidy). After having exhausted all empirical methods sometimes it is indicated to consider referring the couple to another center. As a last resort there is the possibility to discuss gamete donation or adoption.

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