Recurrent implantation failure – real-life cases

In this webinar, Dr Christos Roukoudis, Gynaecologist & Fertility Specialist at IVF-Spain, Alicante has explained the main causes of recurrent implantation failure and discussed 2 complicated cases and what was done to achieve a healthy baby.

Recurrent implantation failure – definition

Dr Christos Roukoudis started his presentation by defining a failure to achieve a pregnancy in women younger than 40 years old after having transferred at least 3 good quality embryos in both fresh and frozen cycles. Another thing is that most of the time a couple never had a pregnancy. It’s crucial to exclude the condition that doesn’t belong to this spectrum such as advanced maternal age, women after 40, diminished ovarian reserve, low-quality embryos, underlying medical conditions such as severe endometriosis, big fibroids or some polyps and also an insufficiently built up mucosa.

Known causes

• blood clotting alterations such as hereditary thrombophilia, Factor V Leiden, hypertrophy, protein S and C deficiency
• immunological alterations such as the antiphospholipid syndrome, KIR-HLA constellation, Celiac disease
• the uterine factor such as chronic inflammation
• embryo factor, one of the most important reasons and that those embryos that we transfer have the highest quality

With advanced maternal age, the rate of unhealthy blastocysts increases and the risks of implantation not occurring is very high.

Recurrent implantation failure – real-life cases

The first case study is about a couple where the female was 39 years old, her AMH was 0, 8 ng/ mL, which was quite low, and the AFC was 5 follicles. Her husband was a bit older than 43 and had oligoasthenozoospermia. They tried to conceive for 3 years, they have gone through 2 previous IVF cycles where they produced 3 blastocysts, all were of good quality and were transferred, but the result was always negative.

• a 43-year-old woman with a husband, with a low AMH level, never got pregnant, had 3 previous failed cycles, the husband was diagnosed with oligoasthenozoospermia

The first step was to start with a proper investigation, for example, the blood clotting, we did the necessary adjustment and screen for hereditary thrombophilia, which was negative, and protein C and S deficiency also was negative. Then we concentrated on the immune system, we screened the blood for certain antibodies that are characteristic of antiphospholipid syndrome, they were negative, and then we searched for KIR type, it was a BB. Regarding the thyroid investigations, normally, if there is no pregnancy or a patient is not trying to get pregnant, the TSH level at 3.2 m U/l is totally fine, but if the couple wishes to achieve a pregnancy, then the value should be lower than 2.5. We started with L-Thyroxine 25 mg, which is the lowest dosage, and after 3 weeks, we checked this again, and it was okay. We also checked the Karyotype, which was normal.

After investigating the partner, we saw that the volume was um 1.2 ml, according to WHO, the threshold is 1.5 ml, the concentration was 35 M/ml, and the threshold is 50, so it was a bit less, the motility should be 39%, it was 32%, so again it was a bit less than normal, normal-shaped sperm should be not less than 4%, it was 4%, but we knew we could work with it, so it wasn’t a very bad result. The karyotype was also normal.

We investigated the DNA fragmentation, and the apoptosis, both of them were in the normal range. When we have a higher DNA fragmentation, we can use very helpful antioxidants, and we can bring them to the normal range. After all of these diagnostics, we started with the first IVF cycle, we used Gonal-F, which is FSH 150 and Pergoveris, which is FSH and LH, both of these were prescribed for almost 10 days. We also gave the growth hormone called Saizen of 0.1 ml during the stimulation, every second day. We do it, as, from our observations, the implantation rate with a low ovarian reserve can be increased by at least 15%. Orgalutran was also prescribed, and after 10 days, and for triggering, we used Triptofem.

During the egg retrieval, we also did a hysteroscopy and an endometrial biopsy because when we have an implantation failure, it is indicated to do a hysteroscopy. The result was normal, we also combined it during the procedure with endometrium biopsy, and we were able to estimate a few things, for example, the implantation window, and the immunological behaviour of the immune cells inside the lining. In the end, we got 2 blastocysts of good quality. The ERmap is an endometrial biopsy that allows us to find the exact time to transfer 1 of those embryos. We know that during the female cycle, oestrogens are produced by the maturing follicles, which cause the endometrium to grow, the luteal phase begins with the ovulation of the second phase of the cycle, and the corpus luteum produces progesterone which makes the endometrium receptive to the fetus for a certain time. We know that in 7% of the woman, it’s after 5.5 days of progesterone release, in the rest of the women, it can be after 6.5 or 4.5 days, we can find out with this ERmap, and the embryo can be transferred accordingly.

The result was that the patient was receptive after 5.5 days, the biopsy was also fine, and we did an immunological screening that turned okay, as well. After that, the idea was to collect embryos so that we were able to increase the possibility of success. We had 2 embryos, and to have realistic chances for success, we needed more embryos, we agreed to do a second cycle, collect a few more embryos and test them genetically to find the healthy ones and afterwards transfer only chromosomally healthy embryos to increase the outcome.

After a month’s pause, and after that, we gave her a contraceptive pill for 14 days to down-regulate her. After she stopped the pill after 5 days, more or less, she had menstruation, we did a scan, and there were only 2 follicles again. We skipped this cycle and waited for the next cycle, and then we were able to find a good cycle with 7 follicles. We used the same medication as the outcome was pretty good before, after 10 days, we got 3 very good blastocysts. On all the embryos, 3 from this cycle, and 2 from the previous cycle, we performed a PGT-A, and we found that out of those 5 embryos, only 2 were suitable for transfer. We prepared to transfer 1 of those euploid embryos, the endometrium thickness was pretty good at 7.5 millimetres, and the result was positive. It was a spontaneous delivery of a baby boy at 38 weeks.

This proves the importance of embryo banking, it not only allows us to increase the success rate by transferring a genetically normal embryo, but we also must have in mind that the couple is now 2 years older, and the ovarian reserve diminishes even more so if we do a second cycle as the couple would like to have a second child, it would be more difficult to generate healthy blastocyst. We achieved success and patience, and now we are going to prepare the patient for the second transfer with one of her frozen blastocysts.

The next case presented a young couple, a 30-year-old woman with an AMH of 3.5 ng/mL, the AFC was quite good, there were 25 follicles. Her husband’s sperm was of normal quality. They tried to conceive for around 2 years, they did 4 previous cycles, 3 IUIs and 1 IVF with 3 transfers, but all were negative.

• a 30-year-old woman with a husband, good AMH level, good sperm quality, 3 failed IUIs, 1 IVF cycle – all results were negative

The patient had a laparoscopy to rule out any blockage in the tubes, and further investigations such as the blood clotting were negative. We also investigated immunological alterations, such as antiphospholipid syndrome, and all were negative. Regarding the HLA constellation, it showed KIR AA which could be important. We were also able to confirm that the patient had Celiac disease without any symptoms, but the antibodies were elevated. The solution is very easy, it’s a gluten-free diet, and afterwards, the antibodies decreased. When we checked the thyroid, the TSH of 3.2 mU/l was elevated, so we prescribed L-Thyroxine.

The partner’s sperm analysis was normal, we did an investigation for the HLA, which was C1C2, this combination of KIR AA and C1C2 is rather not favourable. We also checked the karyotype on both patients, and everything was okay.

Regarding the HLA combination, the endometrium is home to various populations of white blood cells, among them, there are the NK cells which regulate the growth of the invading fetal cells. Every woman has that, and they shouldn’t be very high or very low. Therefore, it could be a matter of the number of NK cells but also their type. Regarding the type, the NK cells are divided into 2 groups according to the receptors they possess, and those groups are then KIR AA, and KIR BB, each woman has one of those. These certain molecules on the surface of the fetal cells are recognized by the NK cells. Half of those molecules come from the partner and the female. It can be C1 or C2, certain KIR HLA combinations are more favourable for developing pregnancy than others. For example, if a woman is KIR BB, she has NK cells, which are not associated with increased risk regardless of the molecules on the embryo’s surface, so it doesn’t matter if the partner is C1C2, C2C2, or C1C1. The situation is different in women who have NK cells that have an unfavourable correlation with the C2 molecule of male origin and are associated with increased risk of implantation failure, early abortions, and also preeclampsia like in our case. It should be emphasized that this relationship does not make pregnancy impossible in such couples, it is only important to recognize the indications in this case of an appropriate history to initiate an appropriate diagnosis and to adjust the treatment starting from the transfer. It means to transfer only 1 embryo, not to burden the female body with more C2C2 molecules from, for example, 2 embryos.

Having all of this in mind, we performed an IVF cycle where we used FSH Puregon for 11 days, and we triggered with Orgalutran from day 7 and Triptofem. During the egg retrieval, we did a hysteroscopy and the endometrial biopsy, we were able to get 14 eggs, out of them, around 12 were mature, 11 eggs got fertilized in the end, and we had very good embryos. We performed ERmap, and we found out that it was pre-receptive. The immunological testing of IMMAP of the lining revealed serious alterations in the population of NK cells, it was quite low in the ratio of TH1 and TH2 cells. After consulting with the immunologists, we prepared a plan for new modulating therapy, and we used additional medication, in this case, it was hCG, so Ovitrelle. We were also able to rule out chronic endometriosis and see that the microbes inside the lining were also okay. We prepared the lining with estradiol and progesterone, and we did the transfer after 6.5 days of progesterone. The lining was thick enough, it was 8.5 millimetres with 3 layers. We transferred only 1 embryo, we had a positive pregnancy, the patient had a delivery at week 39, and it was a healthy baby girl.

Conclusions

This is another reason for doing proper homework and shows how important it is to check the patients individually and come to the right conclusions so that precious time is not lost. The physician should always initiate the appropriate diagnostic, and individual treatment is extremely important to provide the patients with the highest possibility for success and not do the same thing over and over again.