SCIENCE PULSE    Sperm Preparation Techniques

Human semen is a complex mixture of cells and fluids produced by the various components of the male reproductive system. The objective of sperm preparation is to remove the vigorously swimming sperm from this mixture, leaving behind the dead, dying or otherwise poorly swimming sperm, additional cells, enzymes and other factors that comprise the seminal fluid. A sperm cell is incapable of fertilizing an oocyte until it has separated from the seminal fluid.

We use a variety of separation techniques in the laboratory that are tailored to the procedure that the sperm will be used for, and modified according to the quality and type of sperm sample we receive. The average man manufactures about 250 million sperm in a 24 hour period. From a single ejaculate, we will only use 100,000 sperm for each oocyte that we have to inseminate in an IVF cycle. But for an intrauterine insemination, we want to get as many motile sperm as possible into the female reproductive tract, so we will therefore be using a much higher overall fraction of the sperm. Alternatively, for men who have no sperm in their ejaculate and for whom we have to retrieve sperm surgically from the testicle, we want to biopsy the minimum amount of tissue that will give us one sperm for every oocyte that has to be inseminated.

There are two general methods that we employ for the vast majority of sperm processing in the laboratory. The first is a density gradient centrifugation procedure in which the sperm sample is gently spun through 1-3 columns of a viscous solution of saline coated colloidal silica particles. The layers of silica are created by delicately layering different silica particle densities on top of each other in a test tube, and then layering neat semen on top. This method takes advantage of the fact that living sperm are dense compact cells that pass easily through the columns, while dead or dying sperm that are less dense due to leaky membranes are trapped with other cells and debris in the interfaces between the layers. The second method for preparing sperm takes advantage of the sperm’s natural swimming abilities by placing neat seminal fluid in proximity to some culture medium and allowing the sperm to swim from one to the other. There are many variations in this technique including the swim-up (semen is layered under the medium), or the converse method called the swim-down, and the actual method used depends mainly on the quality of the sperm sample. The swim-up is primarily used for samples that have good numbers of highly motile sperm from which only a small fraction needs to be recovered. The swim-down technique works better when sperm are swimming weakly and need the help of gravity to separate from the seminal fluid. For an individual with vanishing numbers of sperm (say a few hundred) we may use a swim-out technique. Here, the sperm are placed in the center of a small drop of medium and an embryologist will wait with a needle at the edge of the drop, picking up the first sperm to get there. One of the big criticisms of the ICSI procedure, where individual sperm are injected into oocytes, is that the embryologist chooses the sperm. However, with the use of the swim-out procedure, there is some degree of “natural selection” as we choose the sperm that are quickest in getting to the edge of the drop. We also choose sperm that are the normal size and shape, and that are free from defects (such as a bent neck) if we have the luxury to do so. In rare cases we have to use every sperm we have, so there’s no “selection” whatsoever. In most of the cases where we’re processing samples that have normal numbers of sperm, the sperm isolated by density centrifugation or by swim-up will be “washed” once or twice before being introduced to the oocytes. This involves suspending the sperm in a volume of culture medium and then centrifuging gently so that the sperm can be concentrated and removed from the medium, while leaving behind any trace of the silica particles or seminal fluid that may have carried over from the first processing step. Although sperm can be damaged by centrifugation, these steps are necessary to ensure that the sperm are free of contaminants that could prevent fertilization.

There are many other methods used to process sperm samples but we use them so rarely that they are scarcely worth mentioning. For example, samples with a high amount of debris can be filtered through glass wool or processed by sedimentation to clean them up before they undergo any of the procedures already described. In addition, we can treat a semen sample with chemicals in certain situations, but this again only happens under somewhat desperate circumstances. If a semen sample is extremely viscous or clotted, we can digest it using the enzymes amylase or chymotrypsin. If none of the sperm are moving we can treat them with pentoxifylline or caffeine to try to stimulate movement. When performing ICSI, we need to know that sperm are alive, and movement is our primary indicator. We can try to stimulate movement using drugs, but for the sperm that are to be used to fertilize the oocytes, we prefer to go drug-free. Here, we place the sperm into a hypo-osmotic solution (regular culture medium that has more water than normal) and as water enters living sperm their tails coil. These we can then inject into oocytes.

For patients that purchase frozen sperm from a sperm bank, the bank will usually offer the option of buying the sperm processed or unprocessed†. Processed sperm, usually labeled “IUI sperm”, costs a little more since the sperm bank has already prepared it for use. Unprocessed or “ICI sperm” is essentially neat semen that has been frozen. Women who do their own inseminations at home buy this type of sperm and inject it into their vagina after it is thawed. If you buy ICI sperm with the intention of having an intrauterine insemination, we will process the sperm as above to remove the seminal fluid and dead sperm. ICI sperm cannot be placed into the uterus since semen contains many contaminants such as bacteria, but also because semen can cause painful uterine contractions.

On a given day in our laboratory, one embryologist is primarily responsible for processing sperm samples, and each embryologist is assigned to this task about once a week. Each sample has different characteristics and the individual doing the processing must make informed decisions on the best approach for recovering the sperm that we need. It is an interesting and demanding area of the laboratory, but we enjoy the challenge of maximizing the potential of each sample that we receive.   
Joe Conaghan, PhD, HCLD

† For more information on frozen sperm and the products sold by sperm banks, see the “How do I Buy Sperm?” article in the April 2005 newsletter which is available on our website (www.infertilitydoctor.com).

 Joe Conaghan, PhD, HCLD PFC's ART Laboratory Director is internationally recognized for his work with embryos. His background includes involvement in the first PGD on human embryos. His high standards and extensive experience bring national recognition to our laboratory. He also trains fellow embryologists for licensure and is an inspector for CAP, the licensing body for IVF laboratories in the USA.

ASK THE EXPERTS    Blocked Tubes

  Pacific Fertility Center Team
Left to Right: Front: Philip Chenette, MD, Isabelle Ryan, MD, Carolyn Givens, MD
Back: Joe Conaghan, PhD, Carl Herbert, MD, Eldon Schriock, MD

Question: My doctor says I have blocked tubes. What causes this and what treatment options are available?

Answer: A blocked fallopian tube is a common cause of infertility. The fallopian tube is the harvester of the egg, floating over the surface of the ovary, picking up the egg after it is released. Sperm meets the egg in the outer one-third of the tube. If the tube becomes blocked, the egg may not be picked up, the tube may not transport sperm, and pregnancy will not occur.

Tubal blockage can occur from infection, such as chlamydia, gonorrhea, appendicitis, or tuberculosis, from an abnormal pregnancy, an ectopic pregnancy, or from surgery, as in a tubal ligation, when the tubes are intentionally tied to prevent pregnancy.

New easier procedures have been developed to improve pregnancy rates in women with tubal blockage. In the past, surgery was performed to fix the fallopian tubes, but these procedures are now rare. Today, techniques like tubal cannulation for proximal tubal occlusion, and salpingectomy for distal tubal occlusion are more often used. The choice of procedure depends on the location of the blockage.

Proximal tubal occlusion (PTO) is the blockage of the fallopian tube at its connection to the uterus. On a hysterosalpingogram, the uterine cavity can be imaged, but the opening to the fallopian tube appears only as a small dimple. PTO is commonly caused by muscular spasm in response to the test but often is an indicator of inflammation of the fallopian tube.

PTO can be treated with tubal cannulation, in which a small tube or wire is used to open the connection to the tube. This procedure can be done on an outpatient basis, using a fluoroscopy, an x-ray technique, or through hysteroscopy, a minor surgical procedure in which a narrow viewing tube is placed into the uterus for a direct look inside.

Hydrosalpinx is a blockage at the other end of the fallopian tube, the “distal” portion near the ovary. The blockage tends to form a pocket where fluid collects. Hydrosalpinx literally means, ‘water in the tube’. The hydrosalpinx indicates severe damage to the fallopian tube and indicates complete blockage.

Hydrosalpinx creates problems for patients undergoing in vitro fertilization (IVF). These blind pockets of fluid sometimes will leak their contents into the uterus, interfering with embryo implantation. Toxic effects on both the uterine lining and the embryo can result. In patients undergoing IVF, the chance for pregnancy if a hydrosalpinx is present drops by 50%. In addition, there is a higher risk of miscarriage and ectopic pregnancy.

Removal of the fallopian tube is a simple laparoscopic procedure that improves pregnancy rates with IVF. The procedure can be performed in under an hour, under anesthesia, as an outpatient procedure. Pregnancy rates with IVF are dramatically improved after removal of the fallopian tube.

PFC’s doctors are pioneering other techniques for treatment of tubal blockage that do not require surgery, such as Essure. Essure is a small micro-insert that is inserted into the fallopian tube under hysteroscopy. Without using an incision, the problem tube can be treated, and IVF performed.

Thankfully, medical advancements designed to treat blocked tubes have demonstrated significant success, helping many patients have a successful pregnancy when they otherwise might not have.  
 Philip Chenette, M.D.

  

Blocked Tube & Open Tube Pregnancy Rates
Women with blocked fallopian tubes on average have better embryo quality than those with open tubes. Since there is an obvious single barrier to pregnancy, the chance of problems with eggs and sperm is lower. Patients with tubal blockage cannot conceive on their own, but with treatment can carry and deliver a pregnancy at excellent rates.

FROM US TO YOU    New Place, Familiar Face

 Pacific Fertility Center is pleased to announce the arrival (or return, if you will) of Mariluz Branch, Laboratory Supervisor to Pacific Fertility Center’s IVF Laboratory. Mariluz brings over a decade of laboratory experience dating back to the early 1990s when she was an embryologist at the San Francisco Center for Reproductive Medicine (SFCRM), now a part of Pacific Fertility Center. She was there during the infant stages of SFCRM, working with Pacific Fertility Center’s Drs. Herbert and Chenette.

At SFCRM, Mariluz played an instrumental role in developing their IVF lab, and, after 11 years, she traveled abroad for several years, moving to London where she held a similar position at an IVF lab. Although there were differences in the processes of working at an IVF laboratory in the UK, she never lost her passion for embryology, and, after moving back to the states earlier this year, she was reunited with many of her former SFCRM colleagues at Pacific Fertility Center. The motivation to work at Pacific Fertility Center was to work in an environment that had good pregnancy rates, trustworthy staff, and high professional standards. Since being at PFC, she has been very impressed with the laboratory’s knowledge, meticulousness, and commitment to do what’s best for the patient.

“Teamwork is one of our team’s biggest strengths,” said Mariluz. “For example, we have a witnessing system during every critical stage in the laboratory. This allows us to run as safe and secure a lab as possible. We also have a large enough staff to prevent us from being overloaded, which helps eliminate any confusion.”

Mariluz has training in preimplantation genetic screening (PGS) and preimplantation genetic diagnosis (PGD) as well as extensive experience with ICSI, cryopreservation and embryo handling. She will bring this and other expertise to her role, which will consist of conducting clinical research studies with the aim of increasing embryo quality and pregnancy rates, and will eventually be overseeing the PGD program. She is also working on developing oocyte (egg) freezing at PFC through a technique called vitrification (versus the more traditional slow freezing method). Mariluz is enthusiastic about the significant and ongoing advancements in assisted reproductive technology (ART) and is pleased to play a role in helping Pacific Fertility Center’s patients realize their dream of building a healthy family.

CONCEPTION HEALTH    Acupuncture and Infertility

 PFC patients often inquire about the use of acupuncture as a part of their infertility treatment, and, as such, PFC brings you insight from Bethany Richardson M.S., L.Ac and Leslie Oldershaw, L. Ac., two Bay Area acupuncturists who integrate fertility treatment into their practices. As background, Bethany Richardson was originally a massage therapist who took a Chinese diagnosis course while working on her Shiatsu certification. That course sparked her interest in pursuing a degree in acupuncture and led her to discover her love for science and the integration of eastern and western medicine. Leslie Oldershaw entered the field of acupuncture after spending her college years dedicated to pre-med courses. She became aware of Chinese medicine as a system of treatment while in college, and it fit perfectly with her passion for eastern philosophy and culture. She had always been interested in women’s health and found that fertility treatment in Chinese medicine truly encompasses all aspects of a woman’s health. Recently, PFC interviewed Leslie and Bethany about their approach to infertility, integrative medicine, and the strengths and weaknesses of acupuncture as a fertility treatment.

PFC: When a patient first comes to you and cites infertility as the reason for their visit, what sort of initial work-up do you do?

Leslie Oldershaw: I do a very comprehensive intake that involves an interview that takes a full medical history. Depending on their history, they may have had labwork done, or I may be ordering tests. From a fertility standpoint I like to see the basic hormone panels, including a cycle day 3 FSH, TSH, Estradiol, and also a mid-luteal progesterone test. If they have done a Clomid Challenge test, I love to see those results. If they have had a HSG or an ultrasound, I like to see that as well. From a general wellness perspective, I need to see a CBC and lipid panel and a comprehensive metabolic panel. When it comes to age, if a patient is younger we can be less aggressive. If a patient is older, we will be more aggressive. If they are 30, I will do this basic work-up, but I will be more inclined to just look at how they do over the next 6 months. If someone who is 40 comes to me, I will recommend that they consult a reproductive endocrinologist. They may want to have more of an accelerated timeline in terms of their treatment options. One of the challenges that I have is that I get patients who don’t want to do the western tests. They don’t want a workup, or the partner doesn’t want to do a semen analysis. I spend a lot of time educating patients to make an informed decision rather than shooting in the dark. I will be more persuasive with my arguments as the patient gets older!

Bethany Richardson: Normally, by the time a patient sees me they have already been to a fertility expert. I ask them to bring in their most current blood work. If they have had an antral follicle count, I want to know what that is. The basal temperature chart gives me a wealth of information. I look at it more in terms of a Chinese diagnosis, not necessarily are they ovulating or when they are ovulating, which is important, but are they running too hot or too cold. It gives me a lot of subtle information from a Traditional Chinese Medicine (TCM) standpoint. If I am looking at a woman who is younger, maybe 31 or 32 years old, then I look at her history of antibiotic use and if they have a history of digestive problems. I look at her menstrual cycle, her PMS, and if she is able to detoxify her hormones correctly. If I am looking at an older woman, I look and see if she is too hot or too cold. Does she have an excess of hormones? I see people mainly when they are desperate. They come to me after two cycles of IVF and want to do everything they possibly can for the next cycle. And unfortunately, I would be more effective if I saw them earlier on, but I do what I can when patients come to me.

PFC: If a patient comes to you and she already knows she must do IVF or IUI to get pregnant, what type of protocols do you offer in conjunction with her fertility treatment?

Leslie Oldershaw: When I work with a patient leading up to the treatment cycle, there are a couple of different protocols we can utilize. The more comprehensive protocol requires 2-4 months and allows you to incorporate nutrition, acupuncture and herbal modalities. We can do a tremendous amount to build a patient up, particularly if they are coming out of a miscarriage. We also work with patients on a shorter timeline, where they might be coming in to work with us a few weeks before an IVF cycle, and then we are primarily relying mostly on acupuncture to promote good blood flow and circulation. We will talk to the patient about nutritional support, including a pre-natal vitamin and omega-3 essential fatty acids. There is not enough research at this point to use fertility medications in conjunction with herbs. If things don’t go as expected the doctor needs to be able to troubleshoot what went wrong. My feeling is that acupuncture is of benefit to everyone. The people for whom acupuncture does not work are the patients who are very needle phobic. And I have to say, my IVF patients are champs. They look at my very small needles and they scoff!

Bethany Richardson: The type of treatment depends where on the continuum they are. I look at whether or not they have done IVF before, and what the results were. Were they a poor responder? How old are they? Sometimes they come to me and they tell me their IVF cycle is in three weeks. I can’t do anything with herbs in three weeks, but I can do some acupuncture. If it is six weeks or eight weeks, then we will have a real protocol. From my perspective the hormones you inject are a very warm tonic, which can burn out your cooling system. Depending on how they react to the injectables, I might try to work with them to take some time off from IVF and load them up with cooling tonics. What I see then is that cervical mucus increases, sleeping patterns regulate and stress levels go down. It’s hard for me to prove that it works, but I honestly believe that it does. Stress management, diet, acupuncture and IVF combined can add up to a much greater level of success.

PFC: What are the strengths and weaknesses of using acupuncture to treat infertility?

Bethany Richardson: It regulates the hormones, helps detoxify the body so you don’t have systemic inflammation, reduces PMS and pain. I think its only downfall is that it takes time. And a lot of the time—women aren’t willing to wait. And that’s where I come in again and say, it’s not a sprint, it’s a marathon. Acupuncture isn’t a golden pill. If you continue to eat poorly and not give your body good nutrients and antioxidants, then I can’t do as much.

PFC: Is there anything you want to add about treating infertility with acupuncture?

Leslie Oldershaw: I have always been keen on integrative approaches. There is a tremendous amount that western medicine can do to help people achieve a pregnancy. But what makes a critical difference for me is the integrative approach. What I am doing with my patients is different than what a Reproductive Endocrinologist does, but together we can work synergistically to create the best outcome.

Bethany Richardson: I am very excited about the future. I am hoping that eventually there will be research money that is earmarked for this arena. Often times acupuncture is seen as competitive with western medicine, and I don’t think it has to be. If we join forces we can be more effective together.

MIND/BODY@PFC    Saturday Workshop

  Stressed or anxious attempting to conceive?

Attend Mind/Body@PFC workshop and learn healthy,
positive ways to reframe your journey to pregnancy.

Saturday, September 16th from 9AM to 4PM
Call (888) 834-3095 for fee and workshop information and registration forms.

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-- Best regards from all of us at Pacific Fertility Center.