(2002) assessment of post vasectomy semen samples British

doi:10.1136/jcp.55.11.812

2002;55;812-816 J. Clin. Pathol.

P Hancock and E McLaughlin

(2002)

assessment of post vasectomy semen samples

British Andrology Society guidelines for the

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REVIEW

British Andrology Society guidelines for the assessment of

post vasectomy semen samples (2002)

P Hancock, E McLaughlin, for the British Andrology Society

.............................................................................................................................

J Clin Pathol

2002;55:812–816

The British Andrology Society guidelines for the

assessment of post vasectomy semen samples

recommend that initial assessment is undertaken 16

weeks post vasectomy and after the patient has

produced at least 24 ejaculates. The laboratory should

examine a freshly produced seminal fluid specimen by

direct microscopy and if no sperm are seen the

centrifugate should be examined for the presence of

motile and non-motile spermatozoa. It is recommended

that the clinician should give clearance after the

production of two consecutive sperm free ejaculates. In

cases of persistent identification of non-motile

spermatozoa the referring clinician should advise the

patient regarding the cessation of other contraceptive

precautions. Surgeons are responsible both

preoperatively and postoperatively for the counselling of

couples regarding complications and the possibility of

late recanalisation after clearance.

..........................................................................

s a lear ned society, the British Andrology

Society (BAS) has sought to improve

standards of diagnostic andrology services

within the UK via educational courses and the

publication of guidelines for the screening of

semen donors.

In 1993, the BAS conducted a

pilot study within the membership and was

instrumental in the setting up of the UK National

Exter nal Quality Assurance Scheme (UKNEQAS)

for Andrology.

In April 1999, at the ﬁfth meeting

of the UKNEQAS for Andrology user group at

Manchester Royal Inﬁrmary Postgraduate Medi-

cal Centre, the UKNEQAS specialist advisory

group debated with the delegates the problems

associated with the assessment of semen samples

after vasectomy. Over 100 delegates were present

and took part in discussion sessions, and it was

proposed from the ﬂoor and endorsed by all

present that the BAS should produce guidelines

for laborator ies undertaking post vasectomy

semen analysis, in an attempt to promote best

practice in this difﬁcult but medico legally impor-

tant area of laboratory andrology practice.

AIMS

The aims of the BAS guidelines on the assessment

of semen samples after vasectomy are to give

guidance to laboratory staff to ensure standardi-

sation of seminal analysis protocols and reporting

of results. After consultation with members of the

British Association of Urological Surgeons, the

Royal C ollege of Obstetricians and Gynaecologists

(RCOG), and the Family Planning Association

(FPA), the committee and members of BAS have

produced these guidelines. The BAS guidelines do

not deal with the counselling of patients or

discuss the indications for male sterilisation, all of

which are considered in the RCOG guidelines on

male and female sterilisation.

INTRODUCTION

Vasectomy is regarded as one of the safest and

most effective methods of birth control,

and it is

popular particularly among the many stable cou-

ples who have completed their families.

Al-

though vasectomy is a relatively simple proce-

dure, it is not without complications,

and careful

preoperative counselling is required.

Failure to

provide and to document adequate preoperative

infor mation and counselling are common causes

of litigation.

Complications include the short

ter m problems of wound infection, scrotal hae-

matoma formation, and primary surgical failure,

together with the longer term issues of chronic

epididymal pain

and spontaneous reversal.

71112

FERTILITY OF RESIDUAL SPERM POST

SURGERY

Evidence suggests that virtually all ejaculates will

contain potentially fertile spermatozoa immedi-

ately after vasectomy,

and that patients should

continue with contraceptive precautions until

they have been advised by their clinician other-

wise. In the small number of studies performed so

far, the spermatozoa remaining within the male

reproductive tract rapidly become immotile, often

within a few days of the vasectomy operation,

13–15

and usually by three weeks.

Although standardised follow up studies are

scarce,

it has been suggested that several

unplanned pregnancies have occurred immedi-

ately after vasectomy, and these have been attrib-

uted not to technical failure of surgery or

recanalisation, but to the patient failing to comply

with instructions regarding continuing contra-

ceptive precautions.

EARLY FAILURE OF SURGERY

Both technical failure (such as missed vasa defer-

entia) and early recanalisation of the vas deferens

have been documented.

In a large study of

.................................................

Abbreviations: BAS, The British Andrology Society; FPA,

Family Planning Association; RCOG, Royal College of

Obstetricians and Gynaecologists; UKNEQAS, UK

National External Quality Assurance Scheme

See end of article for

authors’ affiliations

.......................

Mr P Hancock, Department

of Microbiology, Yeovil

District Hospital, Higher

Kingston, Yeovil, Somerset

BA21 4AT, UK;

[email protected]

Accepted for publication

26 June 2002

812

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16 796 vasectomy operations carried out within one centre in

the UK, the surgeons reported a failure to achieve sterility in

72 men. This early failure rate was quoted as 0.36%,

and has

been linked to operator experience

819

and the technique used

by the surgeon.

320

Early failures were usually identiﬁed by the

presence of motile spermatozoa in ejaculates examined three

to six months postoperatively,

18 19

and surveys have suggested

that many surgeons will opt to repeat surgery

721

as soon as

possible.

LATE FAILURE

Late failure is deﬁned as the presence of spermatozoa in ejacu-

lates after documented azoospermia in two post vasectomy

semen analyses.

12 22

Although uncommon, the late failure rate

has been reported to be 0.04–0.08%,

18 19

and recanalisation is

usually only discovered after pregnancy in the female

partner.

12 18 22 23

It has been suggested that because women may

not inform their partners of a pregnancy for fear of being

accused of inﬁdelity, but may seek a termination, the late fail-

ure rate may be an underestimate.

Similarly, the identiﬁcation

of spermatozoa in 9.7% of ejaculates from a group of 186 men

undergoing semen assessment prereversal

of their vasectomy

also suggests an under reporting of late recanalisation.

LABORATORY ASSESSMENT OF POST VASECTOMY

SEMEN SAMPLES

Sample collection

The RCOG has recommended that before vasectomy

the

patient should have received information from his clinician

regarding the likelihood of a successful vasectomy operation

and the possibility of recanalisation. It is recommended that

such information should be given both verbally and in writing

and repeated by the clinician at the time of ﬁnal clearance.

The laboratory should ensure that the clinician has clear

written instructions for sample collection (appendix 1) and

suitable specimen containers to give to the patient. Laborato-

ries should advise clinicians that men must be asked to collect

their entire ejaculate by masturbation into the non-toxic sam-

ple container provided.

Semen samples should be collected after an abstinence

period of no less than 48 hours and no more than seven days,

and maintained at body temperature before delivery to the

laboratory (ideally within one hour of production in line with

cur rent World Health Organisation protocols

). It is recom-

mended that laboratories endeavour to examine samples

within four hours of production. If this is not feasible, the

period elapsing between production and examination should

be stated on the report form. However, in cases of the persist-

ent presence of non-motile sperm in the ejaculate, further

fresh samples should be examined. These samples must be

delivered to the laboratory within one hour of production and

again maintained at or near, but not exceeding, body

temperature until receipt in the laboratory. It is suggested that

an appointment be made with the laboratory to ensure that

the sample is examined immediately on arrival, to establish

sper m motility. Postal delivery of samples is not recommended

because sperm motility declines rapidly with time.

“It is recommended that laboratories endeavour to

examine samples within four hours of production”

If, however, there is a signiﬁcant risk of patient non-

compliance or hardship, laboratories may make exceptions

and accept samples by post. Laboratories accepting postal

samples should ensure that patients understand the sample/

request form labelling requirements and the current regula-

tions for the transport and packaging of samples (see HSE

1996). Postal samples must abide by these requirements and

be sent by ﬁrst class post.

In cases of paternity dispute, samples must be delivered

personally to the laboratory and all details conﬁrmed with the

patient before the sample is examined.

Many men will produce ejaculates that contain residual

(usually non-motile) sper matozoa for some weeks after

surgery.

The BAS recommends that patients should be

instructed to ensure that they have had at least 24

ejaculations,

5618

and preferably wait at least 16 weeks before

submitting a ﬁrst semen sample for examination.

27–29

This will

reduce the number of false positive samples and thus

minimise both patient inconvenience and repeat laboratory

assessment.

On delivery of the semen sample to the laboratory

reception, staff should ensure that the collection container is

clearly labelled with at least three patient identiﬁers (such as

name, date of birth, and clinic number) and the corresponding

for m is completed in full with details of sample collection and

abstinence period (appendix 2). If an unlabelled sample con-

tainer is delivered, for medicolegal reasons the laboratory

should not examine the specimen and should request a repeat

ejaculate for assessment.

Patients should be given a further collection container and

instructed to produce a second ejaculate for examination at an

interval of two to four weeks after the initial assessment. In

the event that samples delivered by post contain non-motile

sper matozoa, it is mandatory that a freshly produced sample

be examined within one hour of production to exclude the

possibility that motile sperm are present.

Sample examination

After registration, freshly produced semen samples should be

examined as soon as liquefaction is complete, ideally within

one hour of production,

and preferably within four hours of

production. Before examination, semen samples should be

stored either at room temperature or in an incubator at 37°C.

The specimen should not be subjected to extremes of

temperature (< 20 or > 40°C) because this will signiﬁcantly

impair sperm motility.

The sample should be mixed well and a 10 µl aliquot pipet-

ted on to a clean non-toxic glass slide using a direct displace-

ment pipette and the semen covered with a 22 × 22 mm

coverslip. This gives a suitable depth (20 µm) to allow any

motile sperm present to swim freely.

Using phase contrast microscopy, it is recommended that

the entire (22 × 2 mm) coverslip area should be examined in a

systematic grid search pattern for the presence of sperm. Any

sper m detected should be categorised as either motile or non-

motile and reported to the referring clinician (appendix 3).

If no sperm are observed by direct microscopy, the entire

semen sample should be transferred using a sterile pipette

into an appropriate conical bottomed non-toxic polypropylene

centrifuge tube and centrifuged at no less than 3000 ×g for 15

minutes.

Failure to centrifuge the sample at the appropriate

“g” force may result in the failure to recover spermatozoa from

the ejaculate.

30 31

The entire centifugate or “pellet” should be resuspended in

a minimum volume of autologous seminal plasma (< 100 µl)

and the entire sample examined systematically (as described

above) for the presence of motile and non-motile

sper matozoa.

The BAS recommends that an estimation of

sper m concentration or numbers of spermatozoa observed in

each high power ﬁeld (×400 magniﬁcation) should be

reported to the clinician.

After centrifugation, the pellet may contain a large number

of cells including germ cells, epithelial cells, and leucocytes.

The presence of many cells may make the detection of any

sper m in the pellet very difﬁcult and the laboratory should

advise the clinician that these cells might have obscured any

sper m present (appendix 3). Early examination, before the 16

week postoperative period, may exacerbate this problem

because of the cellular response.

BAS guidelines for post vasectomy semen samples (2002) 813

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In a small minority of cases, when the semen sample is

extremely viscous, laboratories may be unable to obtain a

suitable centrifuged pellet to examine. In this situation, it is

recommended that a repeat sample should be requested. In

cases of persistent seminal viscosity, the entire ejaculate may

be incubated for 30–60 minutes at room temperature with a

protease such as 1% bromelin (Bromelain, CN Biosciences UK,

Beeston, UK). A stock solution of 10% bromelin in phosphate

buffered saline (pH 7.2) should be prepared and aliquots

stored at −20°C until required.

Aliquots can be thawed and

diluted to 1% with phosphate buffered saline and brought to

temperature when required. Incubation with bromelin may

immobilise any motile sperm present and the laboratory

should advise the clinician accordingly (appendix 3).

CLEARANCE

The RCOG and FPA guidelines state that at least two

azoosper mic consecutive samples two to four weeks apart

must be obtained before contraceptive precautions can be dis-

pensed with. Recent studies have re-emphasised this period as

appropriate.

It is the responsibility of clinicians not the labo-

ratory to communicate the results of semen assessments to

their patients.

A large proportion of patients (up to 25%) fail

to submit post vasectomy semen specimens for

assessment,

27 28 33

or fail to comply with instructions regarding

sample collection,

making reliable laboratory assessment

impossible.

Proscriptive sample collection may increase the

risk of non-compliance by patients; however, this should be

minimised by appropriate counselling and laboratory staff

should endeavour to ensure patient compliance by accommo-

dating clinical and patient wishes (such as postal delivery of

semen specimens) whenever possible.

SPECIAL CLEARANCE

Persistent non-motile spermatozoa in the initial two ejacu-

lates is not uncommon, with studies reporting up to 33% non-

azoosper mic samples at three months,

and 10% of ejaculates

containing non-motile sperm at six months.

In one prospec-

tive study, the median time to azoospermia was 10 weeks, with

most men (93%) being azoospermic by week 20.

Similarly,

sper m can reappear in ejaculates 12 months after surgery,

despite previous sperm free ejaculates.

Over 1000 men taking

part in a prospective long term follow up study have submit-

ted semen samples at one, two, and three years post

vasectomy.

Twenty men had a positive semen analysis but

there were no reports of unwanted pregnancies in the

partners of these men.

Discussion in the literature has

suggested that the risk of pregnancy occurring from these

non-motile sperm is small,

and probably no more than the

risk of pregnancy after two azoospermic semen samples, as a

result of spontaneous recanalisation.

12 21

It has been suggested

that repeat ejaculates seven months after surgery are probably

unnecessary because pregnancy is very unlikely to occur.

27 36

“It is the responsibility of clinicians not the laboratory to

communicate the results of semen assessments to their

patients”

Samples in which non-motile sperm are repeatedly detected

should be examined within the recommended time periods to

optimise motility detection. Before the issuing of “special

clearance” the clinician may ask the laboratory to conﬁrm the

vital status of the non-motile spermatozoa using an appropri-

ate staining technique.

If any motile sperm or substantial numbers of immotile

sper matozoa (> 10

) are detected then the laboratory should

infor m the clinician promptly because many surgeons will opt

to repeat surgery

721

as soon as possible.

These men with low numbers of persistent non-motile

sper matozoa in their ejaculates (that is, after seven months

and at least 24 ejaculations) may be given “special clearance”

by their clinician to discontinue other contraceptive precau-

tions following appropriate oral counselling and written

advice (appendix 4) regarding the risk of pregnancy.

21 29 36

The BAS recognises the problems associated with patient

non-compliance in the production and assessment of post

vasectomy samples, as highlighted by various workers.

37 38

Because single, early (< 16 weeks) samples are more likely to

give inconclusive results requir ing further patient samples,

these laboratory guidelines coupled with careful pre vasec-

tomy counselling are designed to ensure that appropriate

clearance should be available to men and their surgeons

within 20 weeks of the operation. The BAS suggests that close

adherence to these guidelines will provide a high quality

accurate laboratory service to patients and clinicians following

vasectomy.

REVISION OF GUIDELINES

It is the intention of the BAS committee that these guidelines

will be reviewed on a regular basis and updated as necessary.

ACKNOWLEDGEMENTS

The BAS thanks Dr H Cooling, Senior Clinical Medical Ofﬁcer in Fam-

ily Planning, United Bristol Hospitals Trust; Professor J Guillebaud,

Medical Director, Margaret Pyke Family Planning Centre London; Mr

C Gingell, Consultant Urological Surgeon, North Bristol NHS Trust

Department of Urology; Mr T Hargreave Consultant Urological

Surgeon, Western General Hospital NHS Trust Edinburgh; Mr C

Parker, Consultant Urolog ical Surgeon, East Somerset NHS Trust; Mr

J Pryor, Consultant Uroandrologist, Lister Hospital London; Dr C

Smith and Dr J Walsh, Clinical Effectiveness Unit, Faculty of Family

Planning and Reproductive Health Care, London; and Dr P Trotter,

Lead Clinician in Contraception and Sexual Health Family Planning

Clinic Taunton and Somerset Hospital for their help and advice.

.....................

Authors’ affiliations

P Hancock, Department of Microbiology, Yeovil District Hospital, Higher

Kingston, Yeovil, Somerset BA21 4AT, UK

E McLaughlin, University of Newcastle, School of Environmental and Life

Science, University Drive, Callaghan, New South Wales 2308, Australia

Take home messages

• This article describes the British Andrology Society (BAS)

guidelines for laboratories undertaking post vasectomy

semen analysis, which are designed to promote best prac-

tice in this difficult but medico legally important area of

laboratory andrology practice

• These guidelines recommend that initial assessment is

undertaken 16 weeks after vasectomy and after the patient

has produced at least 24 ejaculates

• A freshly produced seminal fluid specimen should be exam-

ined by direct microscopy and if no sperm are seen the

centrifugate should be examined for the presence of motile

and non-motile spermatozoa

• Clinicians should give clearance after the production of two

consecutive sperm free ejaculates

• When non-motile spermatozoa are persistently identified,

the referring clinician should advise the patient on the ces-

sation of contraception

• Surgeons are responsible for the counselling of couples

regarding complications and the possibility of late recanali-

sation after clearance

814 Hancock, McLaughlin, British Andrology Society

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Appendix 2 Pathology laboratory request form

Surname ____________________

Forename ____________________

Date Of Birth _____/_____/_____

Address ____________________

Clinic No. ____________________

Requesting Doctor ____________________

Date of Vasectomy _____/_____/_____

Specimen details

When was the semen sample collected? Date ___/___/___

Time ______ a.m./p.m.

Was the whole sample collected? YES/NO

When was the last time you ejaculated before this? Date ___/

___/___ Time ______ a.m./p.m.

Appendix 3 Pathology laboratory report form

Name _______________________

Clinic No. _______________________

Date sample_____ / _____/ _____

Delay to examination ______ hrs

Volume ______ ml

Specimen collection: Complete/Incomplete

Direct microscopy: No sperm seen/____Motile sperm

seen/_____Non-motile sperm seen

Centrifugate: No sperm seen/____Motile sperm seen/

_____Non-motile sperm seen

Vital staining: Live sperm seen/No live sperm seen

Comments:

Centrifugate contained many cells—these may have obscured

any sperm present

Viscous sample treated with bromelin—this may have caused

immotility of sperm present

No further specimens necessary

Repeat sample requested

Clinician to review

Appendix 4 Example letter to patient

Miss Jane Jones Consultant Urologist

Outpatients Clinic, St Elsewhere’s Hospital, Newtown

Mr Smith

12 The Avenue, Newtown

15th September 2000

Dear Mr Smith

This letter is to confirm that there were no active sperm present in your post vasectomy semen samples; however, a very small

number of non-motile (inactive) sperm were still present in your last sample. There is no evidence that a pregnancy has

occurred in men producing these low numbers of inactive sperm.

At your initial appointment we discussed that vasectomy operations are not 100% successful and there is always a small fail-

ure rate of between one in two to three thousand cases. This means that there is a very low but not zero chance that you

might conceive in the future.

If at any time in the future you have any problems regarding your vasectomy operation or there is a possibility that Mrs Smith

has become pregnant, then you must contact you general practitioner immediately.

Yours sincerely

Miss Jane Jones

Appendix 1 Semen analysis (post vasectomy)

Please read these instructions thoroughly

Semen analysis is carried out only by appointment with the laboratory. Appointments can be made by phoning 01234

56789, between 0900 and 1600 hours Monday to Friday. Your first appointment must be at least 16 weeks and 24 ejacu-

lations after your operation.

Collection of the sample

The specimen must be collected in the morning. You must not have had sex or masturbated for 48 hours before collecting the

sample but should have had an abstinence period of no longer than seven days. First wash your hands and genitals with soap

and water, please ensure complete removal of all soap residues and dry thoroughly.

The container

Please use the sterile container provided. Do not open the container until you are ready to produce the sample.

Collect the sample by masturbation, collecting the entire specimen directly into the container. (Do not use a sheath/condom

for collection, as they are harmful to sperm. Please note: No other forms of sample collection are acceptable.)

Seal the container immediately after sample collection with the lid; make sure that the lid is on tightly. Do not use adhesive

tape. Write your name, date of birth plus date and time of production on the container label. Record your period of absti-

nence and the date of your operation on the form provided and ensure the name of the requesting consultant/general prac-

titioner plus your name, address, and date of birth is also stated on the form.

Delivery of the sample

Deliver the sample and completed form to the Pathology Laboratory within one hour of collection. The sample must be

examined before it is four hours old, for this reason postal delivery is not acceptable.

** UNLABELLED SPECIMEN POTS WILL NOT BE PROCESSED **

If you have any difficulties associated with the collection and delivery of your sample please contact the laboratory on tele-

phone number 01234 56789.

BAS guidelines for post vasectomy semen samples (2002) 815

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