In vitro

What is in vitro fertilization?

In vitro fertilization is a reproductive medicine procedure involving the combining of gametes out side of a woman’s body and then implanting the fertilized egg in the uterus.

The first successful in vitro fertilization was performed in 1977 on a patient treated for blocked fallopian tubes. The method has been developed and perfected over the years which has in turn expanded the list of indications qualifying patients for the procedure and minimalized the occurrence of complications and undesirable effects. Currently it is more and more often recommended method of treatment and has the highest success rate of all available assisted reproduction methods.

The in vitro procedure can be divided into the following stages: hormone stimulation; biopsy and oocyte retrieval; fertilization and embryo culture; embryo transfer; cryopreservation of embryos for future use; cryotransfer.

The nOvum Fertility Clinic specializes in IVF treatment of infertility and our average success rate over 10 years of procedures (2004-2013) is 43%.

What are the indications for in vitro?

lack of or inoperable occlusion of fallopian tubes. Changes in the fallopian tubes which make sperm migration and the movement of a fertilized oocyte and embryo through the oviduct difficult or impossible:

  • patients with permanent damage
  • patients who cannot undergo surgical restoration of oviduct functions
  • patients with

when none of the diagnostic tests indicate the reason for the infertility:

  • when the infertility has lasted for longer than 3 years (2 years according to NICE)
  • if the patient is older than 35 a shorter period is also accepted


occurrence of tissue similar to the endometrium outside of the uterus, resulting in mechanical changes, inflammation, bleeding and adhesions in the reproductive system or outside of it:

  • I, II degree after a period of natural attempts like in the case of idiopathic infertility
  • III, IV degree

after 12 stimulated cycles without a pregnancy

Applies to women who:

  • had their ovaries removed (due to cancer or inflammatory issues)
  • premature menopause
  • underwent many failed attempts using their own oocytes
  • have test results indicating a limited number of oocytes


depending on the woman’s reproductive potential insemination or IVF with donor sperm is indicated.

The process of in vitro

Hormone stimulation is intended to stimulate the ovaries to produce ovarian follicles containing oocytes. During each cycle several to over a dozen such follicles are produced.

In most cases patients take contraceptive pills in the cycle preceding ovarian stimulation. This is intended to impair ovarian function and prevent the development of ovarian cysts. Towards the end of the cycle, during a visit combined with an ultrasound scan, a final decision is reached on the application and type of hormone stimulation adopted. Depending on the Patient’s age, their FSH and AMH levels in blood serum and the ultrasound scan results of the ovaries, the doctor will recommend one of the three protocols: long, short, or short with an antagonist.


Suppression of the pitiuary

If the long protocol is selected, as of the 21st to 25th day of the cycle (which is soon after the patient has stopped taking contraceptive pills), the patient starts taking analogues of the hypothalamic hormone (gonadotropin-releasing hormones) which suppress the secretion of the FSH and LH hormones by the pituitary gland. In a natural cycle the FSH and LH hormones stimulate the ovaries to grow the ovarian follicle and mature the oocytes. In a stimulated cycle the doctor is in charge of the amount of hormones affecting the ovaries by “switching off” the pituitary gland and externally administering a controlled amount of hormones. The suppression of the pituitary secretion is confirmed by a test of the LH hormone and estradiol levels in blood, and an ultrasound examination.

In the short protocol the suppression of the pituitary gland does not go as far as in the long protocol.

In the antagonist-assisted protocol the suppression of the pituitary gland is as thorough as in the long one, but it can be achieved much faster (soon after the administration of the antagonist) than is the case in the long protocol.


Stimulating follicle growth

The next stage is the ovarian stimulation to ensure that ovarian follicles grow and contain mature oocytes, and to activate the endometrium to grow, which is necessary for the implantation of an embryo – this stage is common for all stimulation protocols. Gonadotropins are administered via intramuscular or intradermal injections, or so-called pens. Patients who are prescribed pen medications will be additionally trained by a nOvum nurse or midwife on the administration of the drug.

The details of the method and sequence of administering the medications will be provided to patients at the initiation of the stimulation. If you are concerned or in doubt about the method of taking the medicines or the individual stages of the treatment, please contact our consultant.

! It is extremely important that Patients do not alter the doses of the prescribed medications, or the times of their administration.

! As you start taking gonadotropins you will not be allowed to take any medications containing acetylsalicylic acid as this increases the risk of bleeding following the biopsy and may result in a surgical intervention being required.

The hormone therapy is subject to multiple checks by the attending doctor through the use of ultrasound scans and monitoring estradiol levels in blood (on average 3-6 visits will be required during the stimulation). The visits are intended to observe the growing follicles and, if necessary, adjust the amount of gonadotropins administered.

Preparing for the biopsy

The hormone stimulation comes to an end when the ovarian follicles grow to the required size - approximately 17-20 mm in diameter - which usually happens 7-14 days after administering the gonadotropins. When the follicles grow to the required size an injection (hCG) is administered causing the release of the oocytes to the fluid in the ovarian follicles. Approximately 34-38 hours following the injection of the hCG the follicular biopsy will take place to collect the follicular fluid containing oocytes.

! Patients who, based on the ultrasound examination of the ovaries, have more than 10 oocytes retrieved are requested to consider the following:  should all of the oocytes be fertilised. This is a crucial decision to make! The number of the fertilised cells and the potential number of embryos developed should be consistent with the couple’s plans concerning their expected offspring. Too many frozen embryos (for the couple’s procreation plans) can lead to a difficult dilemma for the patients in the future...

! Patients deciding that not all of the retrieved oocytes should be fertilised are also requested to consider donation of the egg cells to those couples for whom this would be the only chance of having their own offspring.

Couples are requested to arrive at the clinic 15 minutes prior to the scheduled appointment. The procedure is preceded by an anaesthesiologist briefing during which the doctor reviews the current health condition of the patient and places a plastic cannula (PVC) used to administer medications or fluids. The PVC will be removed by a nurse shortly before the patient is discharged, following a blood pressure reading, some 3-6 hours after the biopsy.

During that time the patient’s partner masturbates to provide the required sperm sample. The male should have practised sexual abstinence for 3-6 days. The period of sexual abstinence should not be any longer or shorter.

Shortly before the procedure patients are requested to urinate, as a full bladder may be an obstacle to the biopsy.

 The retrieval of oocytes is performed in a gynaecolgical chair in a procedure room (right next to the IVF laboratory), during a short loss of consciousness induced by general anaesthesia. The patient goes under immediately following the administration of the anaesthetic agents - going under is quick and pleasant as is waking up. The procedure lasts approximately 10-20 minutes and consists of puncturing the ovaries through the vaginal vault (under ultrasound guidance) and collecting the follicular fluid with the egg cells. An anaesthesiologist monitors the patient at all times throughout the procedure. The follicular fluid containing the oocytes is delivered to the IVF laboratory directly after the procedure. The patient slowly regains consciousness and remains in the recovery room for about one hour.


After the biopsy Patients remain at the clinic for 3 to 6 hours and during that time they are offered snacks and drinks; they are also free to spend their time at leisure in one of the many relaxation areas across the clinic - the cafeteria, the garden, or a resting area.

Patients with a history of ovarian procedures and those who had more than 15 oocytes collected and residing farther than 50 km from Warsaw are required to stay in Warsaw for at least 24 hours.

Cases of nausea or vomiting following the procedure are very rare. A sensation of thirst and dryness in the mouth is a normal symptom, being a side effect of the medication used. Full sensory and motor functionality is restored within 60 minutes of the procedure. After that patients I allowed solid foods and fluids. Lower abdominal pain may be relieved by taking analgesics. Following the procedure patients should not operate any machinery.

In case of any worrying symptoms (such as strong abdominal pain, pain in the collarbone area, fever (over 38 Centigrade - raised temperature following a procedure and the administration of progesterone is normal), loss of consciousness, bleeding from the genital tract), patients are requested to contact the Clinic immediately, call the alarm number provided after the procedure, or get to the nearest gynaecological ward indicated by the doctor.

Immediately after retrieval the follicular fluid is delivered to the IVF laboratory where a microscope is used to select the oocytes. An oocyte is approximately 0.2 mm in diameter. Together with the cumulus oophorus, or a cluster of granulosa cells providing nutrition to the egg cell, it is visible to the naked eye. Any oocytes found are transferred to special supplements and placed in an incubator.

During that time the sperm provided by the partner in a special container is prepared. The preparation consists of selecting the best structured and the most motile spermatozoa in a special medium using centrifugation techniques.

Some 3-6 hours from the isolation the pan containing the egg cells is combined with the required number of spermatozoa with a concentration of approximately 100,000/ml of the medium, and replaced in an incubator. After the next 18-20 hours, which is usually the following morning, the egg cells are rinsed from the sperm suspension, separated from the layers of the granulosa cells, and checked for fertilisation. A confirmation of fertilisation is the presence of two pronuclei visible under the microscope: male and female (they carry the complete 46 chromosomes- 23 from the mother and 23 from the father) and the presence of a second polar pody produced by the cell after fertilization. Such afertilised oocyte is called a zygote. Approximately 70-80% of egg cells undergo normal fertilisation at this stage.

! The patient is informed about the results of the fertilisation over the telephone on the day following the biopsy, at the time agreed to when she was discharged.

Following an assessment of the fertilisation and a change of the culture media, the zygotes are placed in the incubator again for continued culture. 25 hours following insemination the zygote will divide into 2 daughter cells, or blastomeres, and will become an embryo. 44 hours following the insemination, which is approximately 2 days later, the embryo will feature 4 blastomeres, and after 68 hours, that is on the third day, 8 blastomeres. It is typical to perform the embryotransfer on the 2nd or 3rd day following the biopsy.

Four days after the retrieval the embryo reaches the morula stage. The differentiation between cells at this stage is such that individual blastometers cannot be seen.

On the fifth day the embryo enter the blastocyst stage and the inner cell mass (which will become the fetus) and the surface cells (which will become the placenta) are visible. This is the last stage at which the embryo can develop outside of the mother’s body. This is when the lysis of the zona pellucida takes place, involving the hatching of the blastocyst which is then capable of being implanted on the uterine wall.

In some situations the transfer may be performed on the fifth day following the biopsy, when the embryo reaches the blastocyst stage. Transferring the embryo at this stage can significantly improve the chances of a pregnancy, and that is why only one blastocyst is transferred. Extended culture is offered to patients who have many (more than 8-10) well-developed embryos in the second/third day.

The embryotransfer is routinely performed on the 2nd or 3rd day after the biopsy. At this stage the embryo has 4-8 cells. A thin plastic catheter is used to take a maximum of 2 embryos along with a small amount (0.3 ml) of the culture media and place it in the uterus. The procedure is performed in a gynaecological chair, in the transfer room adjoining the IVF laboratory. The procedure is painless and does not require the use of any anaesthesia. The transfer of the embryo is performed when the patient has a full bladder.

Directly prior to the transfer the couple can watch the embryos on the computer screen in the transfer room, which is next to the IVF laboratory, and following the transfer they, can obtain a picture of the transferred embryos. The partner may be present during the procedure. Some 10 minutes following the procedure the patient can get up from the chair and, having received a medication schedule, leave the clinic.

The embryotransfer may be performed under ultrasound guidance or not (the so-called 'clinical touch', i.e. no ultrasound guidance is used). Currently most transfers in nOvum are performed under ultrasound guidance, however it is not always necessary. Our experience, as well as scientific research (“Human Reproduction” in 2008) shows that an effective embryotransfer which results in a pregnancy, when performed by an experienced doctor, does not require the use of ultrasound guidance.

Two embryos are usually transferred, although recently in order to minimise the risk of multiple pregnancies, especially in young patients, single embryos are transferred. The remaining embryos are cryopreserved to be used if the first attempt is unsuccessful, or if you decide to have another baby. On the day of the transfer you will be asked to sign an embryo cryopreservation agreement with a storage term of 12 months after the biopsy. If the embryos are not used over that time, the agreement needs to be extended.

We recommend that patients take their time with returning to their daily routines after the transfer- detailed information will be provided in the discharge summary. If medically justified or when the patient’s occupation involves a significant amount of manual labour, patients will be issued doctor’s certificate indicating that they are not fit for work for a specific period of time.

The implantation of the embryo in the uterine cavity is supported through the administration of hormones which positively affect the process. Starting after the retrieval of the egg cells (biopsy) until approximately the 10th-12th week of the pregnancy, or – in the case of a failure - until the next menstruation, vaginal suppositories containing progesterone will need to be administered. This is the so-called supplementation of the luteal phase and is intended to allow the implantation of the embryo. In a natural cycle progesterone is secreted by the corpus luteum developed after the ovarian follicle ruptures. In an IVF cycle, as a result of multiple ovarian follicles being ruptured, the developing corpora lutea may be inefficient and fail to produce the sufficient amounts of progesterone, which is especially important in the long protocol over the first 2 weeks after the transfer - hence the need for an external supplementation of the hormone.

If the implantation of the embryo proceeds normally, then after 14-17 days from the embryotransfer a hormone pregnancy test will provide positive results.

Please confirm that you are pregnant by filling in the form I am pregnant.

If the test confirms a pregnancy the patient continues to be given progesterone until the completion of the first trimester (10 - 12 weeks from the IVF procedure) - at that time the dose will be adjusted based on the available test results. An ultrasound examination confirming a pregnancy is performed twice: on the 25th day of the procedure to determine if the gestational sac is to be found in the uterus, and on the 35th day of the transfer to confirm the foetal heart rate. If the examination is performed at nOvum this will be the last visit with the attending doctor in charge of the treatment. nOvum offers continued pregnancy care, but only with an obstetrician specialising in antenatal care. If a pregnancy has been confirmed by a clinic other than nOvum, we request that you submit the relevant information to us- preferably by filling in the form I am pregnant.

[Translate to Angielski:] Jeśli test potwierdza ciążę, pacjentka kontynuuje leczenie progesteronem aż do zakończenia I trymestru ciąży (10 - 12 tygodni od przeprowadzenia zabiegu IVF) - w tym czasie dawka leku zostanie skorygowana w zależności od aktualnych wyników badań. Badanie USG potwierdzające ciążę wykonuje się dwukrotnie: w 25. dniu po zabiegu w celu ustalenia czy pęcherzyk płodowy znajduje się macicy, a kolejne, w 35. dniu po transferze, w celu zarejestrowania czynności serca płodu. Jeśli badanie wykonywane jest w nOvum, jest to ostatnia wizyta u lekarza prowadzącego dotychczasowe leczenie.

Leczenie zakończone upragnioną ciążą to zawsze ogromna radość rodziców i satysfakcja dla lekarza prowadzącego. Prowadzenie ciąży w nOvum oczywiście jest możliwe, ale nie zawsze u lekarza, który prowadził leczenie niepłodności. Wiemy, że pacjentki, które przez czas leczenia związały się ze swoim lekarzem przyjmują z żalem tę informację, lecz taka jest konieczność ze względu na znaczną liczbę par oczekujących na leczenie niepłodności. Jednocześnie lekarze opiekujący się pacjentkami ciężarnymi w nOvum to specjaliści z wieloletnim położniczym doświadczeniem klinicznym i praktyką w opiece nad kobietami, które zaszły w ciążę po leczeniu niepłodności.

Pierwsze tygodnie: radość i lęk

U wielu pacjentek początkową wielką radość z powodu potwierdzenia dawno oczekiwanej ciąży zastępuje niepokój i natrętnie powracające pytanie: czy wszystko będzie dobrze? Pierwsze tygodnie to często intensywne wsłuchiwanie się we własne ciało i nadmiernie lękowe reakcje na każdy podejrzany objaw. Trudno się temu dziwić, zważywszy jak długą i bolesną drogę przebyli rodzice. Warto jednak pracować nad tym, żeby po okresie trudnego leczenia czas ciąży był radosny i spokojny. Opieka doświadczonego lekarza, ginekologa - położnika, daje poczucie bezpieczeństwa i szybciej pozwala uzyskać równowagę emocjonalną przyszłej mamie. Lekarze opiekujący się ciężarnymi w nOvum rozumieją doskonale wzmożony niepokój pacjentek i poświęcają tyle uwagi i czasu, ile potrzeba na rozwianie wszelkich wątpliwości.

Ciąża to nie choroba - nawet po zapłodnieniu in vitro

Nawet po zastosowaniu najbardziej skomplikowanych technik wspomaganego rozrodu, przebieg ciąży i porodu może być absolutnie prawidłowy i fizjologiczny. Niepłodność jest chorobą, ale ciąża uzyskana w wyniku jej leczenia wcale nie! To, jak przebiega może zależeć od przyczyny niepłodności, ale jeśli nie występują komplikacje, należy prowadzić normalny tryb życia, oczywiście stosowny dla kobiety spodziewającej się dziecka. Nie ma konieczności leżenia, rezygnowania z pracy, z umiarkowanej aktywności fizycznej czy współżycia. Wszystko zależy od samopoczucia kobiety i wyników aktualnych badań. Większość naszych mam, po pierwszych, czasem trudnych tygodniach odzyskuje spokój i radość.

Ciąża podwyższonego ryzyka

Jednak bywa i tak, że ciąża po leczeniu niepłodności wymaga szczególnego traktowania. Wynika to miedzy innymi z faktu, że leczeniu niepłodności często poddają się kobiety po 35. czy nawet 40. roku życia. Naturalna wydolność i sprawność organizmu spada wraz z wiekiem, oczywiście u każdego w różnym stopniu. Ciąża - nawet samoistna - w tym wieku stwarzać może większe zagrożenie cukrzycą ciężarnych, nadciśnieniem indukowanym ciążą i innymi tego typu powikłaniami.

Kolejnym powodem może być ciąża mnoga. Noszenie jednocześnie dwójki lub więcej dzieci stanowi większe obciążenie dla matki. Częściej w takich przypadkach mamy do czynienia z cukrzycą ciężarnych, nadciśnieniem indukowanym ciążą, nasilającymi się obrzękami i żylakami, przedwczesnym porodem dzieci ze zmniejszoną masą ciała. W ciąży bliźniaczej trzeba liczyć się z częstszymi badaniami ginekologicznymi i badaniami USG oraz z koniecznością spędzenia pewnego czasu w szpitalu, zwłaszcza w końcowych jej tygodniach. Ułożenie dzieci uniemożliwiające poród drogami natury, często skłania lekarzy do zakończenia ciąży cesarskim cięciem.

Termin porodu

W przypadku ciąży po zapłodnieniu in vitro termin wyliczamy nie na podstawie daty ostatniej miesiączki, tylko daty punkcji jajników i pobrania komórek jajowych. Od tego dnia do teoretycznego terminu porodu dodaje się 38 tygodni.

Badania prenatalne

Dzięki wczesnej diagnostyce ultrasonograficznej wykonywanej w okresie między skończonym 9 a 13 tygodniem ciąży + 6. dniami, skojarzonej z biochemicznym badaniem próbki krwi pobranej od matki, można ustalić ryzyko wystąpienia niektórych zaburzeń genetycznych płodu, przede wszystkim trisomii chromosomów 21, 18 i 13 oraz niektórych wad wrodzonych.

Najczęstszym i najbardziej znanym zespołem wad jest tzw. zespół Downa, czyli trisomia chromosomu 21. Z wiekiem matki rośnie ryzyko wystąpienia tego zespołu u płodu.

Do testu przesiewowego muszą być przeprowadzone następujące badania:

  1. Ultrasonograficzny pomiar niewielkiej przestrzeni płynowej występującej na karku płodu, zwanej przeziernością karku (NT, ang. Nuchal Translucency). Ze wzrostem grubości przezierności karku rośnie ryzyko aberracji chromosomowych i niektórych wad u płodu, np.: wad serca. Badanie musi być wykonane przez doświadczonego lekarza ultrasonografistę na aparacie wysokiej jakości. Podczas badania ultrasonograficznego zmierzone będą wymiary płodu, jego budowa anatomiczna pod kątem możliwych do zdiagnozowania w USG wad płodu, oceniona zostanie również czynność serca.
  2. Oznaczenie poziomu stężeń następujących substancji w surowicy krwi matki:
    • osoczowego białka ciążowego A (PAPP-A, ang. Pregnancy Associated Plasma Protein A),
    • hormonu beta-hCG (ang. free beta human Chorionic Gonadotropin).
    Ocena zmiany ich stężeń służy do obliczenia ryzyka wystąpienia aberracji chromosomowych. Dane o poziomie hormonu i białka oraz pomiary uzyskane z USG są, po uwzględnieniu wieku matki i informacji z wywiadu, przeliczane na ryzyko dotyczące badanej ciężarnej.

Jak interpretować wyniki testu złożonego USG + PAPP-A + beta hCG?

Test złożony (zwany czasami w skrócie po prostu testem PAPP-A) jest badaniem przesiewowym o bardzo wysokiej czułości wykrywania zespołu Downa, która wynosi ok. 90%, to znaczy, że wykrywanych jest 9 na 10 chorych płodów.

W około 5% przypadków test może być zinterpretowany jako nieprawidłowy i w tej grupie mieści się około 90% płodów z zespołem Downa. Nieprawidłowy (czyli dodatni) wynik testu nie oznacza, że w danym przypadku mamy do czynienia z chorym płodem, a tylko, że badana ciąża zaliczona jest do grupy o podwyższonym ryzyku. Za pomocą testu nie może być postawiona ostateczna diagnoza, konieczne jest wykonanie badania inwazyjnego.

Z drugiej strony prawidłowy (czyli ujemny) wynik badania nie daje gwarancji niewystąpienia u płodu aberracji chromosomowych, oznacza tylko, że szansa na ich wystąpienie jest mała.

Wykluczenie aberracji chromosomowych może nastąpić tylko w wyniku bezpośredniego badania chromosomów pochodzących od płodu, uzyskanych drogą amniopunkcji, kordocentezy lub biopsji trofoblastu, które należą do badań inwazyjnych.

Nie stwierdzono znacząco częstszego występowania wad płodu po zastosowaniu technik wspomaganego rozrodu. Częstość występowania wad płodu jest minimalnie większa niż w populacji niemającej problemów z płodnością, dla której ryzyko to wynosi ok. 3%, i, według oficjalnego stanowiska ESHRE (Europejskiego Towarzystwa Rozrodczości Człowieka i Embriologii) zawartego w "ESHRE positionstatement. By SQUART, 07 April 2009' Bitrh defects In IVF children.", wynosi  4,2 - 4,5% (podobnie dla dzieci urodzonych po konwencjonalnym zapłodnieniu pozaustrojowym, jak i po mikroiniekcji ICSI, jak i po spontanicznym zajściu w ciążę po długim okresie oczekiwania na nią). Ten nieznaczny wzrost jest związany z niepłodnością, a nie technikami jej leczenia.

Według Bonduelle i Van Steirteghema badających dzieci urodzone w latach 1991 - 1999 po zapłodnieniu metodą ICSI, nieznacznie (do 1,56%) wzrasta ryzyko aberracji chromosomowych (zmiany w liczbie chromosomów) 1. W ogólnej populacji to ryzyko wynosi 0,5%. Może mieć to związek z większą częstością występowania aneuploidii (nieprawidłowej liczby chromosomów) w plemnikach niepłodnych mężczyzn.

Dlatego w przypadku pacjentek powyżej 37. roku życia, a także u wszystkich pacjentek po mikroiniekcji ICSI, zaleca się wykonanie badań prenatalnych (amniopunkcji) między 12. a 16. tygodniem ciąży. Polegają one na nakłuciu macicy przez skórę i pobraniu wód płodowych do badania genetycznego. Badanie jest inwazyjne, toteż nie wszystkie pary wyrażają zgodę na jego przeprowadzenie.

Istnieje również możliwość wykonania w Przychodni Lekarskiej nOvum nieinwazyjnych, przesiewowych badań prenatalnych w kierunku zespołu Downa i otwartych wad układu nerwowego, tzw. Double test (dawny TRIAS) lub PAPP-A. Zarówno Double test, jak i test PAPP-A określają prawdopodobieństwo występowania wyżej wymienionych wad u płodu. Jeżeli prawdopodobieństwo jest duże, trzeba wynik ten zweryfikować za pomocą amniopunkcji.

All viable embryos created in the IVF laboratory that were not transferred to the uterus will be frozen for later use. This allows patients who want to achieve another pregnancy or for whom the first transfer failed to avoid hormonal stimulation and biopsy- it reduces the strain on the woman’s health and the treatment costs and is thus a very important stage of the in vitro procedure.

Embryos to be used at later stages of the treatment, like sperm and oocytes, are cryopreserved in liquid nitrogen. This is currently the only known method to ensure their preservation.

Contemporary embryology makes use of several methods of cryopreservation, of which two are most commonly used, slow freezing and vitrification which involves rapid freezing in a small amount of fluid. The best option is always selected by the biologist in charge of a procedure. As is the case with any stage of the treatment, there is no single best method which would apply to all patients - this is why, as much as you trust your doctor in selecting the best procedure for you, we also ask that you trust the biologist in selecting the best method of cryopreservation.

One of nOvum’s successes in cryopreservation is the birth of a healthy baby girl after almost 11 years of the embryo being stored at the clinic. In 2000 one of the infertile couples underwent in vitro fertilization. After the procedure the patient was in danger of developing ovarian hyperstimulation syndrome so the doctors decided to cryopreserve all viable embryos. When the patient’s health allowed for a transfer the first pregnancy from the frozen embryos was achieved. In 2001 a baby boy was born and 10 years later on the 16th of October 2011 his twin sister was born- conceived on the same day but waiting for her birth in nOvum for almost 11 years…

After having their first child the parents had to postpone expanding their family for health reasons. However, as soon as it became possible the couple asked for another embryo which had been waiting in nOvum’s embryo bank, frozen in liquid nitrogen, for all these years. The embryo showed life signs after thawing, was transferred to the uterus and a pregnancy was achieved. A healthy baby girl was born in term and received 10 points on the Apgar scale on the day of her birth. 

A Guarantee of Safety at nOvum

One of the most important expectations couples being treated for infertility have, is the assurance that the gametes and embryos they entrust to the in vitro laboratory and the cryo-bank will be always correctly identified. From the moment the egg cells and sperm cells are collected, they are under the care and supervision of the embryologists. This is a great responsibility and requires the best and most efficient quality control system, as well as identifying samples at every stage of laboratory processing. This applies to both the samples collected before fertilization and identifying the embryos while they are being grown in the IVF laboratory, during transfer, while they are being prepared for cryopreservation, while they are stored and during cryotransfer. In the past years nOvum has worked with an original system of multiple checks, however we have always kept an eye out for new technological developments in this area. In 2015 we made the strategic decision to introduce an electronic and independent control system that allows us to keep track of samples at each stage of the laboratory processing. It is a significant undertaking, both from an organizational and financial perspective, which we embarked on keeping in mind the safety of the procedures that take place at nOvum, the comfort of the patients who entrust their biological samples to us, and the working conditions of embryologists who can dedicate their full attention to the medical procedures.

The system we introduced is RI Witness, used in the world’s best in vitro laboratories in 22 countries to supervise over 500 000 in vitro cycles. 

  • Each couple receives a card with a unique, personal code at the beginning of treatment.
  • Every piece of laboratory equipment used during the process of collecting and transferring biological material (e.g. containers for collecting semen samples, laboratory glassware etc.) are tagged with the same code.
  • The ID tags and the electronic code identification system (RFID technology) allow the identification, tracking and registration of the patients’ biological material during each stage of the in vitro fertilization.
  • Each work area and workstation in the IVF laboratory receives the wireless signal sent out by the ID tags, ensuring the safe tracking of the entire cycle. This means that each stage is monitored and registered by a system independent from our internal laboratory protocols.
  • RI Witness confirms the identity of the biological material repeatedly and at every station, only when the identity is confirmed does the system allow for the work to proceed.
  • In the unlikely event that the system registers any potential identity incompatibility of the cells or embryos, the laboratory procedures are automatically blocked by the system, which prevents any potential mistakes. In such a case, a procedure to investigate the incompatibility is immediately launched.
  • The certainty that each stage of the fertilization process in the nOvum laboratory is safely monitored; guaranteeing that the biological material is correctly and exactly identified at each stage of the laboratory procedures.
  • The assurance that you are being treated at a clinic which successfully performs a great number of in vitro procedures, and for which your security and the successful outcome of the treatment are the highest priorities. nOvums philosophy is based on ensuring the highest standards, quality control and the careful supervision of each stage of treatment.
  • The knowledge that your gametes and embryos are cared for by a team of embryologists working in comfortable conditions, which allow them to perform each procedure with absolute care and attention.
  • The certainty that your gametes and embryos are in a secure location, under the constant supervision of embryologists, cryobiologists and efficient electronic systems.
  • The certainty that during the time that your embryos and gametes are cryopreserved, they are tagged and identified through a unique system- they are safe!

Link to a video containing information about the RI Witness system:

Procedures which improve in vitro fertilization success rates

ICSI (Intracytoplasmic Sperm Injection) is an IVF procedure which entails the insertion, using a micropipette, of a single sperm directly to the inner part of the oocyte.

Indications for ICSI:

  • the man’s semen features a relatively small number of spermatozoa or they have impaired motility
  • when the past attempts at in vitro fertilisation resulted in failures (no fertilisation was achieved)
  • the number of the oocytes isolated is small (<5)
  • the patient suffers from endometriosis or unexplained infertility (idiopathic ).

After fertilization through ICSI the rest of the treatment is the same as in the case of classic IVF.

In the event of azoospermia, which involves a lack of measurable level of sperm in the semen, male epidydymis or testicle may be aspirated and, if live sperm is found, microinjected to the oocytes. These methods, abbreviated to PESA and TESE, M-TESE, make fatherhood possible in situations when until recently this was not a valid option.

PESA (Pericutaneus Epidydymal Sperm Aspiration) - a small needle is inserted through the skin of the scrotum to collect sperm from the epididymis.

TESE - Testicular Sperm Extraction- is the process of extracting sperm from the testicle.

M-TESE - Micro- Testicular Sperm Extraction- involves the extraction of tissue fragments during an open testicle biopsy under the guidance of a microscope to obtain spermatozoa for IVF.

One sample collected during the procedure is observed under a microscope to determine presence of spermatozoa, a second sample is sent out for histopathology testing. 

The remaining material- regardless of whether spermatozoa were found - is frozen so that we know whether there is a chance to find spermatozoa and if ovarian stimulation is indicated. When the histopathology confirms there is a chance to find sperm the patient is qualified for ovarian stimulation and IVF.

On the day of the ovarian biopsy, after oocytes are retrieved, the samples from the aspiration/biopsy are thawed in search of live spermatozoa to be used in fertilization.

When performing pericutaneus or microsurgical epididymal sperm aspiration (PESA, MESA) or testicular sperm aspiration or extraction (TESA, TESE), the collected sperm is delivered to the IVF laboratory. This is where spermatozoa are identified which can be used for the IVF-ICSI protocol. If the collected tissue lacks spermatozoa, the extraction or aspiration procedures may be repeated - even many times - but this, however, is not a valid option due to the patient’s stress and the fact that any subsequent (≥3) aspirations or extractions usually result in an almost zero success rate.

In the case of M-TESE the testicular tissue is collected under the guidance of a microscope in order to precisely identify the spots from which the vas deferens potentially containing spermatogenesis will be removed. This is why M-TESE is a much more precise procedure than PESA, MESA, TESA or TESE. 

IMSI (Intracytoplasmic Morphologically Selected Sperm) involves the injection of spermatozoa selected under high magnification into the oocyte. It is a less frequently used variation of ICSI involving the selection, under high magnification (approximately X1000 optical or X6000 digital) of a single sperm to be used for fertilisation. This magnification allows to evaluate the detailed structure of sperm and select the specimens which are almost perfect, based on theory morphology and the criteria established by the method’s creator (B.Bartoov) that is the presence, or its lack, of a vacuole in the area of the nucleus. For now the vacuole’s negative impact on the resulting embryo and subsequent pregnancy has not been proven. This method does not have a proven higher success rate that ICSI. There are research paper which confirm its higher effectiveness but also papers which do not confirm it is more effective than standard ICSI. There are no established indications and recommendations for IMSI. Due to this fact nOvum’s internal criteria for IMSI are:

  • male infertility where a lack (0%) or very low percentage (below 1%) of viable sperm is determined through regular or computer analysis
  • a lack or low percentage (less than 30%) of blastocysts determined in prolonged embryo culture
  • recurring (three or more) miscarriages after excluding other causes
  • recurring (three or more) unsuccessful implantation of embryos after excluding uterine causes

In Vitro Maturation is used in women whose ovarian structure prevents the use of hormone stimulation, making it dangerous in terms of excessive follicles developing and a risk of overstimulation. 

In these patients immature egg cells are retrieved earlier than usual from unstimulated ovaries or ovaries that have only been minimally stimulated. The cells retrieved are immature and cannot be fertilised. They are placed in special supplements containing hormones which in the classic in vitro are used for ovarian stimulation. As a result, on the day following the biopsy (28-32 hours of maturation in an incubator) some 40-70% of the oocytes can be fertilised using intracytoplasmic sperm injection (ICSI). Once fertilisation has taken place the following stages of treatment correspond to the ones applied for the classic in vitro. The chances of achieving a pregnancy are reduced by some 10-20%, but the risk of complications is also reduced for susceptible patients.

Not all cycles are suited for a biopsy, collection and maturation of egg cells. The final qualification is confirmed on the third day of the cycle based on the estradiol levels in blood serum and an ultrasound scan.

Preparing for an IVM consists mainly of monitoring the cycle and observing the follicular growth, as well as checking the estradiol levels. The biopsy is performed between the 7th and the 10th day of the cycle when the follicles, the endometrium and the estradiol levels reach the required values. On the day of the biopsy 10 ml of the patient’s blood will have to be collected to prepare the serum to be added to the maturation media.

The preimplantation genetic diagnosis of the first polar bodies (Polar Body Biopsy) in the oocyte helps enhance the chances of a normally developing pregnancy and birth of a healthy baby for couples who decided on in vitro.

The polar body is a small external fragment of the egg cell developing as a result of its maturation and used by the cell to get rid of any excessive chromosomes. Based on a test indicating the number of chromosomes in the polar body we are able to, to a high level of probability, assess the number of chromosomes in the oocyte.

Each human cell (except for mature reproductive cells) contains, under normal conditions, 46 chromosomes (structures built from DNA holding genes). Mature reproductive cells, such as oocytes and sperm, hold 23 chromosomes each so that a new organism can have 46 after they are combined. Initially, a maturing 46-chromosome oocyte during the so-called meiotic cell division develops polar bodies (first and second) which are used to get rid of the 23 chromosomes, making space for the sperm’s genetic material. Under natural circumstances, after this important function has been executed, polar bodies become fragmented and ultimately vanish. Under laboratory conditions it is possible to screen the polar bodies without harm to the egg cells which will indirectly serve as a way to evaluate the genetic material of the mother cell as the number of chromosomes in the first polar body should be consistent with the number of the chromosomes in the oocyte. These tests cannot, however, rule out any potential genetic disorders passed down from the mother organism, but can, to a high level of probability, rule out the most common ones connected with chromosomal aberrations (number anomalies).

Preconception genetic diagnosis (PCGD) versus preimplantation genetic diagnosis (PGD).

The preimplantation genetic diagnosis (PGD) is a diagnosis of an existing embryo for genetic features. If the embryo is found to be defective, it will not be transferred to the uterus. This help to avoid a genetically risky pregnancy.
However, this does not counteract the development of defective embryos.

For some patients who, because of their medical conditions, can expect their offspring to be genetically affected, the preimplantation genetic diagnosis may be difficult to accept from an ethical perspective. Selecting existing embryos based on their genetic defects is a method that has been used worldwide for years, but continues to be controversial, both from the ethical and medical perspective.

Out of respect for those of our Patients who might struggle with such a dilemma, nOvum decided to apply genetic diagnosis at an earlier stage, the so-called preconception genetic diagnosis (PCGD) and fertilise only those oocytes which carry the normal number of chromosomes.

Indications for polar body biopsy.

The biopsy is indicated whenever there is reasonable evidence that the offspring may be affected by a trisomy (three chromosomes instead of two, e.g. Down syndrome, Edwards syndrome, Patau syndrome) or monosomy (one chromosome instead of two), as well as other genetic conditions which are related to the abnormalities of the egg cell. Affected previous children of the mother, numerous failures of IVF, miscarriages, age of 35 or over are all reasons to consider polar body biopsy. Delayed child-bearing will not change the tough rules of biology - in women aged 40 and over approximately 70% of the oocytes are chromosomally abnormal.

Oocyte diagnosis.

Once retrieved from the ovaries, oocytes are transferred to the IVF laboratory. Mature oocytes are then used to isolate the polar body which is subjected to a genetic diagnosis. The examination takes approximately 6-7 hours, and is a time-consuming process for both the IVF and genetic laboratories. Following the procedure the staff will advise Patients on the number of normal oocytes qualified for fertilisation - only such egg cells will be fertilised. The following stages of the procedure are consistent with the process adopted for the typical in vitro fertilisation making use of intracytoplasmic sperm injection (ICSI), embryo development under laboratory conditions, and transfer of the embryos to the uterus of the future mother.


Under the current state of the art polar body biopsy may increase the chances of a normally developing pregnancy achieved as a result of in vitro fertilisation. This is accomplished thanks to the elimination of genetically abnormal oocytes which, on fertilisation, would become a source of genetically abnormal embryos, and this in turn could result in an implantation failure, miscarriage or birth of a child with a Patau, Edwards or Down syndrome. 95% of genetically defective embryos do not normally develop, resulting in implantation failures or early miscarriages.

Method limitations

This diagnostic method allows to probe chromosomal anomalies only in relation to selected chromosomes in an egg cell. These include the following chromosomes: 13, 16, 18, 21, 22 and X, with a sensitivity of 90-95%.

Embryo culture mediums (like EmbryoGen) prepare the embryo for implantation and contain natural cytokine growth factors GM-CSF. It is present in the woman’s body and is an important regulator of the embryos development process, cell cleavage and impacts embryo survival rates. This means it has a huge impact on achieving reproduction success in women who have lost pregnancies in the past.

A GM-CSF deficiency during pregnancy can lead to irregular placenta development and functioning, which may lead to miscarriage. Healthy women with appropriate GM-CSF production the levels regularly increase during pregnancy, women who have suffered one or more miscarriage might have a GM-CSF deficiency.

Long-term research in over a dozen clinics on a large group of over 1300 patients have shown a 44% increase in successful implantation of embryos created in vitro among women who previously suffered miscarriages.

Enriching the IVF medium with the growth factor brings the medium’s composition closer to the embryo’s natural environment and improves its chances of development.

Indication for use:

  • one or more miscarriage in patient history, after excluding other causes
  • one or more biochemical pregnancies in patient history

It is a medium (nourishment) prepared for embryo transfers which contains hyaluronic acid which helps the binding of the embryo to the endometrium and increases the chances of implantation.  Many research papers investigating whether hyaluronic acid added to the transfer medium does in fact improve the chances of pregnancy have been published. At nOvum we routinely use a medium containing hyaluronic acid for the transfer of most non-frozen embryos. The indications for the use of this medium are established individually (some cultures require different mediums). 

In nature the activation of an egg cells is a series of processes taking place in the cell after fertilization which begin its further division. In a laboratory the activation of oocytes involves placing them (directly after ICSI) in a special medium containing calcium ionophores. Ionophores are molecules capable of, among other things, transporting ions (in this case calcium ions) across the cell membrane. The purpose of using this medium is increasing the concentration of calcium ions inside the oocyte which mimic the natural process which happens in a cell after fertilization (sperm entry) induced by the activating factor in the sperm. Assisted oocyte activation is used for patients in who’s cases previous ICSI attempts did not result in fertilization or had a low (less than 30%) fertilization rate which can be the result of, among other things, a deficiency of the activating agent in the sperm. Using assisted oocyte activation in the case of a zero or low fertilization rate, regardless of the cause, allows the rates to increase (according to many studies and our experience) to around 60-70%.

In the case when a certain number of live, though immotile, sperm have been detected, the sperm can be used for ICSI after being placed in a special medium which activates immotile sperm - this way the embryologist can determine which sperm is alive and can be used for oocyte fertilization. However, one must remember that the chances of the procedure being successful when originally immotile but activated with a special medium sperm is used are lower than in the case of fertilization with properly motile sperm.

Assisted “hatching” of the embryo is a procedure involving the cutting (mechanically, by laser or chemically) the layer surrounding the embryo in a spot where there is the least risk of damaging it.

The layer disintegrates on its own around 5 to 6 days after fertilization which causes the embryo to "hatch" and allows it to implant itself in the endometrium. It has been hypothesised that repeated failed implantations of healthy embryos might be caused by irregularities in the zona pellucida’s dissolution.

The Cochrane analysis researching the effectiveness of AH based on 31 randomized clinical studies with the participation of 5728 patients, did not find statistical confirmation of a higher number of children born after AH in comparison to patients with whom AH was not used. However a no statistical increase in the number of pregnancies has been observed in patients after a few failed transfers. Further research is indicated to confirm these initial observations.

World literature reports cases of monozygotic multiple pregnancies occurring more often following the procedure. We have to take into account the higher risk of a monozygotic multiple pregnancy which, in the case of a multiple embryo transfer, might lead to an overly numerous pregnancy (the risk of one embryo splitting in two and the other also implanting might result in triplets and if both embryos split – quadruplets).

Cases in which the doctor might consider cutting the zona pellucida

  • patient is over 39 years old
  • elevated FSH hormone levels marked on the 1-3 day of the cycle
  • unusually thick zona pellucida (based on the assessment of the embryologist in the IVF laboratory.  In such a case the embryologist informs the patient of any additional indications for AH during a routine phone call the day after the biopsy. Since the cut is made immediately preceding the transfer the decision can be made at the last moment during the viewing of the embryos before they are placed in the transfer catheter).
  • multiple (at least three) failed embryo implantations

We should indicate that nOvum does not routinely perform AH in cases of well-developed (expanded) blastocyst (expansion grade 4 or higher) because an expanded blastocyst has a very thin zona pellucida (otherwise it wouldn’t have expanded so much) and there is no reason to cut it since the probability of the zone disintegrating correctly is high.

The embryoscope is the highly specialized equipment of the nOvum IVF laboratory which allows us to observe the embryo from conception, through all the stages of its development, to the transfer without the need for the embryos to ever leave the incubator and be exposed to temperature and pH changes.

Development and observation of embryos at the IVF laboratory

As soon as the collected gametes, i.e. oocytes and sperm, are delivered to the laboratory, the developing embryo becomes the subject of continuous care of an embryologist. The success of the entire IVF procedure is very much dependent on whether the embryos show the required growth potential, in other words whether they are capable of the correct divisions, implantation in the uterus, and starting a healthy pregnancy. 

At the IVF laboratory we are able to monitor all of these development stages which would normally take place inside a mother’s womb. Directly after fertilisation the oocytes are placed in incubators which offer optimal conditions for the development of embryos, being the exact equivalents of the conditions found inside a woman’s body.

As a standard procedure, once a day embryos are removed from the incubators and viewed under the microscope. Such assessments are usually not performed more frequently as embryos should not to be exposed to changing conditions of the environment in which they develop. 

The assessments of the embryos are intended to determine their growth potential. Embryologists will monitor the following aspects: speed and sequence of cells division, their size and appearance, as well as the degree of fragmentation. Embryo fragmentation is the proportional relation between the correctly shaped cells (which will continue to divide creating new blastomeres) and the cells which split into smaller, nucleus-free parts. If the latter ones account for more than 20% of the embryo, the likelihood of obtaining a pregnancy grows smaller.

Currently nOvum has access to a method allowing for the precise monitoring of the embryos’ development- the embryoscope.

Continuous, non-invasive monitoring of embryos

The embryoscope allows for the continuous monitoring of embryos from the moment of fertilisation, through all stages of their development, to the transfer without the need for the embryos to ever leave the incubator and be exposed to temperature and pH changes. A camera placed inside the incubator takes photographs of the embryos every 20 minutes. The images are collected by the computer, which allows a detailed record covering the entire embryo culture period. An analysis of the recording enables the laboratory personnel to select only the most promising embryos for the first transfer. Others (if any) are frozen for potential future transfers. 

The use of the embryoscope provides the most information when embryos are cultured until they reach the blastocyst stage (over 5 days). However, even shorter observations provide valuable insights into the dynamics and normality of the first divisions, enabling us to offer a prognosis on a potential pregnancy. 

The method is mainly recommended for couples with a higher number of embryos that can be cultured until the blastocyst stage, in the case of a serious male factor which might impede the embryos’ development after the third day, if such problems (impeded development) have already occurred during culture for previous attempts, with a high number of fertilized oocytes to help choose the best embryo for transfer.

At nOvum it is the embryologist who establishes the indicators for culture in the embryoscope based on the factors mentioned above, taking into account that one embryoscope has room for embryos from 6 patients, assuming none of them have more than 12. 

Freezing sperm for oncological patients or patients qualified for IVF who will not be present on the day of the ovarian biopsy.

Information for patients giving semen for freezing and later use:

  • Register for freezing semen through the IVF laboratory Monday through Friday from 9 a.m. to 10 a.m.
  • In order to deposit semen the patient needs to come to the andrology laboratory between Monday to Friday from 8 a.m. to 1 a.m.

Before depositing sperm you should abstain from sexual activity for 2 to 7 days.

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