Q. How can I be sure that PFC will not accidentally confuse my eggs and my husband’s sperm and our embryos with someone else’s?

A. PFC recognizes that even with the best intentions, human error can occur. We’ve therefore designed our strict SurTransfer^SM laboratory security system of color-coding and clearly labeling all specimens and verbally identifying all patients. We have also devoted considerable time and effort into assembling one of the most highly trained teams in the country. Each of our Embryologists is Board Certified and Licensed, even though the State of California does not currently require licensure for Embryologists.
When a patient is scheduled for a procedure, a written procedure requisition is sent by the Physician to the laboratory staff, giving them at least 24hour notice and clear instructions on what is to be done. Each patient is assigned a specific color for their test tubes and Petri dishes; no two patients having procedures on the same day will be assigned the same color. Each of the patient’s specimens is carefully labeled with clear and unique identifying information that includes the patient’s name and date of birth.

During their stay in the lab, eggs, sperm and embryos are kept in incubators. We avoid assigning two cases to a single incubator on the same day. Each incubator has an exterior door and an interior door. Both doors are clearly labeled with name and color code. This labeling protocol allows the embryologist to verify the name twice before ever handling the specimen.

We have two embryologists performing all critical procedures to ensure accuracy; generally one handles the material while the other observes and verifies. We are not required to assign two people to procedures, but redundancy eliminates the possibility of an error.

Both embryologists sign off after checking the paperwork, labeling the specimen and performing the procedure.
Accepting sperm samples: When a man delivers his sample, we require it to be labeled with his unique information, including name, birth date and signature. We ask to see identification. The embryologist receiving the sample will sign that s/he received it and note the time and date of receipt. If s/he passes the sample to another member of staff, that individual will sign for it, thus continuing a chain of responsible custody.

Egg retrieval: A patient undergoing egg retrieval is asked in the retrieval room to identify herself before receiving sedating drugs. The embryologist will not rely on the physician, nor state the patient’s name and ask for a “yes or no” answer, but will instead ask her to state her full name. This avoids any possible miscommunication. As the procedure gets underway, two embryologists will take responsibility for accepting the collected eggs.

Inseminating eggs: This is arguably the most important part of the IVF procedure. While it is a relatively simple procedure to perform, we are sensitive to its significance. Without any exceptions, two embryologists perform the insemination. Even if there is only one egg to inseminate, or even if there is only one insemination on a given day, two people do it.

Embryo transfer: Similar to the retrieval procedure, one embryologist will ask the patient her name and a second embryologist will witness and verify that the correct embryos are loaded into the transfer catheter. As a final check, the embryologist will hand the catheter to the physician and state the patient’s full name and the number of embryos.

Freezing and thawing of sperm or embryos:

Frozen specimens are extensively labeled and catalogued. Thawing can only be directed by a physician, and as a rule an embryologist never handles or thaws a specimen without a witness. Once a specimen is thawed, there’s no going back.

1. Embryos are stepped through increasing (freeze) or decreasing (thaw) antifreeze concentrations in a 4-well plate.	2. Once saturated with antifreeze, the embryos are loaded into small straws.	3. The straws are placed in a controlled- rate freezer.
4. Once frozen, the embryos are placed in state-of-the-art, computer controlled storage tanks.	5. The straws snap into labeled metal canes. They are barely visible through the mist of the cooling nitrogen gas.	6. After thawing, it is not unusual to see one or more dead cells in an embryo. Arrow is to 1dead cell in 4-cell embryo.

To successfully freeze, or cryopreserve any cell, its water must first be removed. Otherwise, the water expands into ice crystals as it freezes, and this process bursts or kills the cell. To maintain embryo viability during cryopreservation, the embryo is bathed in antifreeze, which draws the water out of the cells while the antifreeze enters. (1) Made with propylene glycol supplemented with sucrose, the antifreeze is not harmful in any way, provided the embryos are kept cool.

After this first step, the embryos are loaded into small straws (2) and placed in a controlled-rate freezer, which cools the embryos at a rate of -0.3°C/minute until they reach a temperature of -38°C. (3) Then they are placed in, and stored in liquid nitrogen at -196°C. (4,5)

To thaw the embryos, the straw(s) are first warmed to room temperature and the embryos unloaded into a Petri dish. Then they are stepped through decreasing concentrations of antifreeze until eventually all the antifreeze has been removed and the embryos have been rehydrated. (1)

There is tremendous variability in how well embryos tolerate the freeze/thaw procedure, although surprisingly, it has little to do with the quality of the embryos at the time of freezing. A poor quality embryo will endure the process just as well as a good quality one. However, since poor quality embryos have a very low possibility of implanting, they are often not worth freezing.

While every care is taken to protect the embryos during the process, some embryos will have one or more burst cells (6) after they have been thawed. This cell loss results either from puncture by tiny ice shards around the embryo or from rupture as water rapidly enters the cell during thawing.

In 2003, over 80% of embryos survived freezing and thawing with one or more cells intact. On average, 2 out of every 3 cells in an embryo tolerated the process, and we consider an embryo with 50% or more of their cells surviving as having a normal chance of implanting after transfer. If an embryo thaws with fewer than 50% of its cells alive, we usually recommend thawing another embryo if one is available. At PFC 66% of embryos thaw with 50% or more of their cells intact.

After my husband and I learned that we had no chance to become pregnant by natural means, we began to investigate IVF/TESE (sperm obtained by biopsy of the testes) with ICSI (Intracytoplasmic sperm injection) as a way to realize our dream of starting a family. We expected the procedures to be challenging to our bodies, minds, and finances.

We were also concerned about the frequency of twin and triplet births with IVF. As much as we hoped to have a child, we wanted to do everything we could to provide the best start for our child-to-be. We wanted to optimize our chances for a healthy full-term singleton pregnancy, natural childbirth, and breastfeeding, if we could become pregnant.

Dr. Carolyn Givens patiently answered our many questions about IVF and embryo cryopreservation and supported us when we made a choice that was quite unusual at the time: we requested that only one embryo be placed in my uterus during the IVF cycle and that any remaining embryos be frozen. I was 34 at the time and had never been pregnant.

Eight-cell embryo

We had the exceptional fortune that our first IVF/ICSI cycle in August of 1997 produced 13 beautiful embryos, and our transfer of a single fresh 3-day-old embryo during that cycle resulted in the birth of our son Benjamin nine months later.

I was still breastfeeding Ben in 2001 when we decided to try for a second pregnancy. Dr. Givens transferred a single 8-cell frozen embryo during an unmedicated natural cycle. We had explained to Ben that there was a little, little baby in Mommy’s tummy that we hoped might grow to be his brother or sister. About a week after the transfer, Ben said, “Mommy, the little, little baby in your tummy is crying.” A few days later, my period began, and I felt like crying too.

The next month, Dr. Givens transferred another frozen embryo, also without medication. Ben thought this embryo was happy, and he was right: she grew to be his sister Charlotte.

When we were considering the choice to have our embryos transferred one at a time, we were glad to learn that the expense of frozen embryo transfers was only a small fraction of that for the IVF/ICSI procedures. I found embryo transfers performed by Dr. Givens to be gentle and comfortable. Dr. Givens’ respect for our individual preferences made our infertility treatments a very positive experience. Our children have brought us unimaginable happiness.

– Camille, Redwood City

Most couples going through IVF or frozen embryo transfer choose to transfer at least two embryos in order to improve the chances of conception with any one embryo transfer procedure. As Camille’s story indicates, however, in younger patients with nice embryo quality and overall good chances for success, electing to transfer a single embryo is a viable option to avoid the risks of multiple gestation pregnancy. It also illustrates the benefits of embryo cryopreservation for having more than one child with a single IVF stimulation cycle.

– Carolyn Givens, MD

Did you know that 343 babies were born as a result of assisted reproduction procedures performed at Pacific Fertility Center in 2002? Of these 343 babies, 98 or 28.6%, were babies conceived after having been stored as a frozen embryo.

This year marks the 20th anniversary of the first successful birth that resulted from human embryo cryopreservation. Since then, thousands of babies have been born worldwide after having undergone embryo cryopreservation. Freezing of excess good quality embryos allows for the transfer of fewer embryos in the stimulated IVF cycle and therefore ensures fewer high-order (triplets or more) multiple births. This process provides patients with a “back-up” should the initial fresh embryo transfer not result in a pregnancy. It is a much lower cost procedure than starting IVF all over again and often is performed with minimal medications. Frozen embryo transfers (FET) have allowed many of our patients to achieve more than one pregnancy from a single cycle of ovarian stimulation.

How safe is embryo cryopreservation? Even after 20 years, there are few studies in the scientific and medical literature concerning outcomes after embryo cryopreservation. However, the few studies that have been published are thus far reassuring.

The four largest studies performed on children conceived after embryo freezing have been done in England, France, Greece and Sweden. In all of these, the authors reported no significant difference in minor or major malformation rates in babies compared to fresh IVF embryo transfers or spontaneously conceived babies. The largest was the Swedish study, which followed 255 children born after embryo cryopreservation up to 18 months of age. Researchers compared them to 255 children born stimulated IVF cycle and therefore ensures fewer high-order (triplets or more) multiple births. This process provides patients with a “back-up” should the initial fresh embryo transfer not result in a pregnancy. It is a much lower cost procedure than starting IVF all over again and often is performed with minimal medications. Frozen embryo transfers (FET) have allowed many of our patients to achieve more than one pregnancy from a single cycle of ovarian stimulation.

How safe is embryo cryopreservation? Even after 20 years, there are few studies in the scientific and medical literature concerning outcomes after embryo cryopreservation. However, the few studies that have been published are thus far reassuring. The four largest studies performed on children conceived after embryo freezing have been done in England, France, Greece and Sweden. In all of these, the authors reported no significant difference in minor or major mal-formation rates in babies compared to fresh IVF embryo transfers or spontaneously conceived babies. The largest was the Swedish study, which followed 255 children born after embryo cryopreservation up to 18 months of age. Researchers compared them to 255 children born. The longest-term follow-up was conducted in the French study, which followed 82 children, aged 1-9, born after cryopreservation as embryos. The total malformation rate was 3.4%. Incidence of medical and surgical illness was not excessive and scholastic performance in the older children was as expected.

What is the longest time an embryo can remain frozen and still be viable? Just this month, a clinic in Israel reported the birth of healthy twins from a transfer of frozen-thawed embryos that had been cryopreserved for 12 years. Embryos, once frozen, may have unlimited potential for viability, as long as they remain at the extremely low temperatures of liquid nitrogen storage.

At Pacific Fertility Center, we are very proud of our record of success with frozen embryo transfers. We see many healthy children at baby visits that were once stored at PFC as frozen embryos. We believe that the data on safety is reassuring. We see cryopreservation as yet another way for patients to achieve healthy pregnancies through assisted reproduction.

References:

Postnatal growth and health in children born after cryopreservation as embryos. Wennerholm UB, Albertsson-Wikland K, Bergh C, Hamberger L, Niklasson A, Nilsson L, Thiringer K, Wennergren M, Wikland M, Borres MP. Lancet. 1998 Apr 11;351(9109):1085-90.

Perinatal outcome and follow-up of 82 children aged 1-9 years old conceived from cryopreserved embryos. Olivennes F, Schneider Z, Remy V, Blanchet V, Kerbrat V, Fanchin R, Hazout A, Glissant M, Fernandez H, Dehan M, Frydman R. Hum Reprod. 1996 Jul;11(7):1565-8.

Minor congenital anomalies, major congenital malformations and development in children conceived from cryopreserved embryos. Sutcliffe AG, D’Souza SW, Cadman J, Richards B, McKinlay IA, Lieberman B. Hum Reprod. 1995 Dec;10(12):3332-7.

Follow-up of children conceived from cryopreserved embryos. Sutcliffe AG. Mol Cell Endocrinol. 2000 Nov 27;169(1-2):91-3.

Outcome in children from cryopreserved embryos. Sutcliffe AG, D’Souza SW, Cadman J, Richards B, McKinlay IA, Lieberman B. St Mary’s Hospital, Manchester. Arch Dis Child. 1995 Apr;72(4):290-3.

Pregnancy and child outcome after assisted reproduction techniques. Tarlatzis BC, Grimbizis G. Hum Reprod. 1999 Sep;14 Suppl 1:231-42.

Twin delivery following 12 years of human embryo cryopreservation: case report. Revel A, Safran A, Laufer N, Lewin A, Reubinov BE, Simon A. Hum Reprod. 2004 Feb;19(2):328-9.